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PMID:4209 | Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. | The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 degrees C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not beta-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5-9.5 at 30 degrees C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 degrees C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/mo, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized. | Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 degrees C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not beta-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5-9.5 at 30 degrees C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 degrees C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/mo, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized. | [
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PMID:4210 | Biosynthesis of chloramphenicol in Streptomyces sp. 3022a. Identification of p-amino-L-phenylalanine as a product from the action of arylamine synthetase on chorismic acid. | Products obtained from the action of arylamine synthetase on [G-14C]chorismic acid were fractionated by gel filtration and ion exchange column chromatography to yield a partially purified radioactive component with an arylamine function. From its ultraviolet absorption spectrum and thin-layer chromatographic behaviour the product was considered to be p-aminophenylalanine and the identification was confirmed by co-crystallization with an authentic specimen. Specific deamination of the product with L-amino-acid oxidase indicated that it was the L-epimer. These results strengthen previous evidence that arylamine synthetase is at a branch point in the shikimic acid pathway, specifically diverting intermediates to the synthesis of chloramphenicol. | Biosynthesis of chloramphenicol in Streptomyces sp. 3022a. Identification of p-amino-L-phenylalanine as a product from the action of arylamine synthetase on chorismic acid. Products obtained from the action of arylamine synthetase on [G-14C]chorismic acid were fractionated by gel filtration and ion exchange column chromatography to yield a partially purified radioactive component with an arylamine function. From its ultraviolet absorption spectrum and thin-layer chromatographic behaviour the product was considered to be p-aminophenylalanine and the identification was confirmed by co-crystallization with an authentic specimen. Specific deamination of the product with L-amino-acid oxidase indicated that it was the L-epimer. These results strengthen previous evidence that arylamine synthetase is at a branch point in the shikimic acid pathway, specifically diverting intermediates to the synthesis of chloramphenicol. | [
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PMID:4211 | Uptake and efflux of succinic acid by uninduced mycelium of Claviceps purpurea. | Claviceps purpurea PRL 1980 grew on partially dissociated succinic acid (pH 4) but not on fully dissociated succinic acid (pH 7.2). Myeclium suspended in 42 mM solution of partially ionized succinic acid (pH 4; 60.1% nonionized, 39% monoanion, and 0.9% dianion, K+ salt) over a period of 25 min accumulated more succinic acid carbon than mycelium suspended in highly ionized solution (pH 6.8; 0.01% nonionized, 4.8% monoanion, and 95% dianion). The greater accumulation from partially ionized solution was not attributable solely to metabolism of succinic acid nor to the lower external concentration of potassium ion. Rate of uptake by sodium azide and iodoacetate-treated mycelium was proportional to external concentration at least up to 200 mumol/ml. External potassium or sodium ion was not required for uptake by inhibited or uninhibited mycelium and external sodium ion and glucose did not allow concentration of succinic acid. The internal concentrations of succinic acid carbon expressed as succinic acid in cell water were about the same as the external concentrations. Uptake was not appreciably affected by extent of ionization of external succinic acid but accumulation was markedly affected. A plot of accumulated succinic acid carbon against external pH produced a bimodal curve with the two maxima corresponding to the maximal concentrations of nonionized and monoanion succinic acid. The bimodal curve probably results from overlapping of two separate curves; the nonionized form accumulating efficiently because of one interaction with the cell and the monoanion form accumulating efficiently because of another interaction. Uptake from concentrated solution is by diffusion and efflux is rapid but not complete. Efflux is not retarded by presence of phosphate in the external solution. | Uptake and efflux of succinic acid by uninduced mycelium of Claviceps purpurea. Claviceps purpurea PRL 1980 grew on partially dissociated succinic acid (pH 4) but not on fully dissociated succinic acid (pH 7.2). Myeclium suspended in 42 mM solution of partially ionized succinic acid (pH 4; 60.1% nonionized, 39% monoanion, and 0.9% dianion, K+ salt) over a period of 25 min accumulated more succinic acid carbon than mycelium suspended in highly ionized solution (pH 6.8; 0.01% nonionized, 4.8% monoanion, and 95% dianion). The greater accumulation from partially ionized solution was not attributable solely to metabolism of succinic acid nor to the lower external concentration of potassium ion. Rate of uptake by sodium azide and iodoacetate-treated mycelium was proportional to external concentration at least up to 200 mumol/ml. External potassium or sodium ion was not required for uptake by inhibited or uninhibited mycelium and external sodium ion and glucose did not allow concentration of succinic acid. The internal concentrations of succinic acid carbon expressed as succinic acid in cell water were about the same as the external concentrations. Uptake was not appreciably affected by extent of ionization of external succinic acid but accumulation was markedly affected. A plot of accumulated succinic acid carbon against external pH produced a bimodal curve with the two maxima corresponding to the maximal concentrations of nonionized and monoanion succinic acid. The bimodal curve probably results from overlapping of two separate curves; the nonionized form accumulating efficiently because of one interaction with the cell and the monoanion form accumulating efficiently because of another interaction. Uptake from concentrated solution is by diffusion and efflux is rapid but not complete. Efflux is not retarded by presence of phosphate in the external solution. | [
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PMID:4212 | Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis. | Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown. | Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis. Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown. | [
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PMID:4213 | Beta-glucanases in the yeast Cryptococcus albidus var. aerius. Production and separation of beta-glucanases in asynchronous cultures. | beta-Glucanases were detected in cell-free extracts of the yeast Cryptococcus albidus var. aerius when grown on glucose as the sole carbon source. The production of beta-glucanases was followed in log-phase cells and stationary-phase cells; the maximal production of beta-(1 leads to 3) and beta-(1 leads to 6) glucanases takes place respectively in log-phase and stationary-phase cells. The results show that there are marked differences in the elution profiles on Sephadex G-50 of fractions containing beta-glucanase from cells grown for 12, 24, 48, 72, and 96 h. The possibility either of replacement changes in fractions containing beta-glucanase activity or of a different synthesis of each beta-glucanase during the growth of the yeast is discussed. The results suggest that all fractions containing beta-glucanases hydrolyze both beta-(1 leads to 3) and beta-(1 leads to 6) linkages. Evidence in support of the conclusion that a low molecular form of beta-glucanase has a molecular weight of 2100 +/- 100 is also shown. | Beta-glucanases in the yeast Cryptococcus albidus var. aerius. Production and separation of beta-glucanases in asynchronous cultures. beta-Glucanases were detected in cell-free extracts of the yeast Cryptococcus albidus var. aerius when grown on glucose as the sole carbon source. The production of beta-glucanases was followed in log-phase cells and stationary-phase cells; the maximal production of beta-(1 leads to 3) and beta-(1 leads to 6) glucanases takes place respectively in log-phase and stationary-phase cells. The results show that there are marked differences in the elution profiles on Sephadex G-50 of fractions containing beta-glucanase from cells grown for 12, 24, 48, 72, and 96 h. The possibility either of replacement changes in fractions containing beta-glucanase activity or of a different synthesis of each beta-glucanase during the growth of the yeast is discussed. The results suggest that all fractions containing beta-glucanases hydrolyze both beta-(1 leads to 3) and beta-(1 leads to 6) linkages. Evidence in support of the conclusion that a low molecular form of beta-glucanase has a molecular weight of 2100 +/- 100 is also shown. | [
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PMID:4214 | Temperature-pH effect upon germination of bacterial spores. | Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed. | Temperature-pH effect upon germination of bacterial spores. Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed. | [
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PMID:4215 | Effect of variou cultural conditions on the fatty acid and lipid composition of Choanephora cucurbitarum. | The fatty acid composition of the total and polar lipid fractions of Choanephora cucurbitarum grown under different cultural conditions were analyzed by thin-layer and gas-liquid chromatography. It was observed that temperature, age, pH, and light influenced the degree of unsaturation, this being due mainly to changes in the gamma-linolenic acid concentration. The conditions used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20-C26) beyond gamma-linolenic acid (C18:3) as reported earlier using growth media containing glutamic acid. The fatty acid pattern of lipid fractions though the same qualitatively, differed quantitatively. The polar lipid fractions, phosphatidyl choline, phosphatidyl ethanolamine, and diphosphatidyl glycerol showed an appreciable variation in gamma-linolenic acid content under different cultural conditions. The degree of unsaturation of the various lipid fractions decreased with increases in temperature, light intensity, and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The significance of these observations is discussed. | Effect of variou cultural conditions on the fatty acid and lipid composition of Choanephora cucurbitarum. The fatty acid composition of the total and polar lipid fractions of Choanephora cucurbitarum grown under different cultural conditions were analyzed by thin-layer and gas-liquid chromatography. It was observed that temperature, age, pH, and light influenced the degree of unsaturation, this being due mainly to changes in the gamma-linolenic acid concentration. The conditions used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20-C26) beyond gamma-linolenic acid (C18:3) as reported earlier using growth media containing glutamic acid. The fatty acid pattern of lipid fractions though the same qualitatively, differed quantitatively. The polar lipid fractions, phosphatidyl choline, phosphatidyl ethanolamine, and diphosphatidyl glycerol showed an appreciable variation in gamma-linolenic acid content under different cultural conditions. The degree of unsaturation of the various lipid fractions decreased with increases in temperature, light intensity, and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The significance of these observations is discussed. | [
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PMID:4216 | Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly. | The cell envelope of Sporillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical deffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH (pH less than or equal to 4.6 dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed. | Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly. The cell envelope of Sporillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical deffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH (pH less than or equal to 4.6 dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed. | [
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PMID:4217 | Premature labour. | Prematurity is by far the commonest cause of neonatal morbidity and mortality. The management of premature labour is empirical because little is understood about the mechanism of labour. Effective uterine relaxant drugs have an important, albeit minor role. Phototherapy has reduced the complications of neonatal hyperbilirubinemia, and the beneficial effect of antepartum corticosteroid therapy in minimizing the risk of respiratory distress syndrome is now convincing. Prophylactic antibiotic therapy in premature rupture of the membranes does not alter perinatal mortality, although postpartum maternal morbidity is reduced. The introduction of neonatal intensive care units has improved the survival rate of premature infants. Sound clinical judgement remains the mainstay in the management of premature labour. | Premature labour. Prematurity is by far the commonest cause of neonatal morbidity and mortality. The management of premature labour is empirical because little is understood about the mechanism of labour. Effective uterine relaxant drugs have an important, albeit minor role. Phototherapy has reduced the complications of neonatal hyperbilirubinemia, and the beneficial effect of antepartum corticosteroid therapy in minimizing the risk of respiratory distress syndrome is now convincing. Prophylactic antibiotic therapy in premature rupture of the membranes does not alter perinatal mortality, although postpartum maternal morbidity is reduced. The introduction of neonatal intensive care units has improved the survival rate of premature infants. Sound clinical judgement remains the mainstay in the management of premature labour. | [
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PMID:4218 | Liver metastases found by follow-up of patients operated on for colorectal cancer. | One hundred and twenty-six patients earlier operated on for colorectal cancer were followed-up once yearly with serum screening tests. The activities of alkaline phosphatase (AP) and gammaglutamyltranspeptidase (GT) were recorded. 58 patients had positive tests. The majority of the patients with liver metastases (20/21) was possible to encircle with these simple serum tests. 38 of the 58 "screening positive patients" were further investigated with celiac angiography and/or liver scintigraphy and liver metastases were very suspect in 29 of these patients. 18 of them were laparotomized and the suspicion was verified in 8.7 of these patients could be subjected to surgery against their liver tumours and 2 of them have then survived more than 2 years. The authors suggest a follow-up system with shorter interval between the examinations. | Liver metastases found by follow-up of patients operated on for colorectal cancer. One hundred and twenty-six patients earlier operated on for colorectal cancer were followed-up once yearly with serum screening tests. The activities of alkaline phosphatase (AP) and gammaglutamyltranspeptidase (GT) were recorded. 58 patients had positive tests. The majority of the patients with liver metastases (20/21) was possible to encircle with these simple serum tests. 38 of the 58 "screening positive patients" were further investigated with celiac angiography and/or liver scintigraphy and liver metastases were very suspect in 29 of these patients. 18 of them were laparotomized and the suspicion was verified in 8.7 of these patients could be subjected to surgery against their liver tumours and 2 of them have then survived more than 2 years. The authors suggest a follow-up system with shorter interval between the examinations. | [
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PMID:4219 | Carcinoembryonic antigen and phosphohexose isomerase, gammaglutamyl transpeptidase and lactate dehydorgenase levels in patients with and without liver metastases. | Plasma carcinoembryonic antigen (CEA) and serum enzyme levels of phosphohexose isomerase (PHI), gamma-glutamyl transpeptidase (psi-GTP), and lactate dehydrogenase (LDH) were measured in 147 patients with malignancy. Levels were higher in patients (particularly with G.I., breast and lung cancers) than in normals or in patients with cancer in clinical remission. Elevations of CEA and of all three enzymes in blood were most frequent in patients with hepatic metastases. CEA elevations correlated directly with PHI levels. Seventy-eight percent of patients with metastatic G.I. cancer could be identified by CEA (greater than 5 ng/ml) alone, as well as 38% with breast cancer and 85% with lung cancer; but only 17% of other cancers could be identified by CEA alone. CEA or one or more enzymes was elevated in 64% of metastatic breast cancer patients, 92% of lung cancer and 41% of other cancers, but enzyme measurement did not increase identification of G.I. cancer over that achieved by CEA alone. These findings suggest that circulating levels of CEA, PHI, psi-GTP and LDH may reflect a direct contribution from the malignant tissue and/or liver malfunction secondary to liver replacement. | Carcinoembryonic antigen and phosphohexose isomerase, gammaglutamyl transpeptidase and lactate dehydorgenase levels in patients with and without liver metastases. Plasma carcinoembryonic antigen (CEA) and serum enzyme levels of phosphohexose isomerase (PHI), gamma-glutamyl transpeptidase (psi-GTP), and lactate dehydrogenase (LDH) were measured in 147 patients with malignancy. Levels were higher in patients (particularly with G.I., breast and lung cancers) than in normals or in patients with cancer in clinical remission. Elevations of CEA and of all three enzymes in blood were most frequent in patients with hepatic metastases. CEA elevations correlated directly with PHI levels. Seventy-eight percent of patients with metastatic G.I. cancer could be identified by CEA (greater than 5 ng/ml) alone, as well as 38% with breast cancer and 85% with lung cancer; but only 17% of other cancers could be identified by CEA alone. CEA or one or more enzymes was elevated in 64% of metastatic breast cancer patients, 92% of lung cancer and 41% of other cancers, but enzyme measurement did not increase identification of G.I. cancer over that achieved by CEA alone. These findings suggest that circulating levels of CEA, PHI, psi-GTP and LDH may reflect a direct contribution from the malignant tissue and/or liver malfunction secondary to liver replacement. | [
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PMID:4220 | Controlled environment culture of bone marrow explants from human myeloma. | Bone marrow biopsy specimens from patients with myeloma were cultured in either 1 of 2 thin-film culture systems, a controlled environment steady state system or a rocker tube configuration of the system, for periods up to 42 days. Both functional and morphological characteristics of the myeloma cells were well-maintained in these systems. Cytocentrifuge preparations of the culture media disclosed hematopoietic cells that included from 5% to almost 100% plasma cells. Histological examination of the cultured specimens disclosed infiltration of the marrow with myeloma cells. Myeloma proteins were released at a steady rate throughout the period of culture after the 1st 4 days. Bone-resorbing activity was demonstrated in the culture media in 7 of 9 myeloma culture media and was well maintained, particularly during the 1st week of culture. This activity was associated with severe osteolytic lesions in the donor patient and marked infiltration of the cultured specimen by myeloma cells. The potential use of these organ culture systems for the further definitive identification of the factor responsible for bone destruction in myeloma is discussed. | Controlled environment culture of bone marrow explants from human myeloma. Bone marrow biopsy specimens from patients with myeloma were cultured in either 1 of 2 thin-film culture systems, a controlled environment steady state system or a rocker tube configuration of the system, for periods up to 42 days. Both functional and morphological characteristics of the myeloma cells were well-maintained in these systems. Cytocentrifuge preparations of the culture media disclosed hematopoietic cells that included from 5% to almost 100% plasma cells. Histological examination of the cultured specimens disclosed infiltration of the marrow with myeloma cells. Myeloma proteins were released at a steady rate throughout the period of culture after the 1st 4 days. Bone-resorbing activity was demonstrated in the culture media in 7 of 9 myeloma culture media and was well maintained, particularly during the 1st week of culture. This activity was associated with severe osteolytic lesions in the donor patient and marked infiltration of the cultured specimen by myeloma cells. The potential use of these organ culture systems for the further definitive identification of the factor responsible for bone destruction in myeloma is discussed. | [
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PMID:4221 | Binding of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to L1210 cell nuclear proteins. | The binding of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) to the proteins of the L1210 cell nucleus has been studied using both [cyclohexyl-14C]CCNU and [chloroethyl-14C]CCNU. Most of the bound [cyclohexyl-14C] moiety of CCNU was found to exist in a form that was stable in acid solution but labile and dialyzable in alkaline solution. A small amount of the cyclohexyl moiety was bound to histones in a stable, nondialyzable form. The drug/protein ratio for the H1 histone was about 0.01 to 0.02 mole/mole. No binding of the cyclohexyl group to acidic proteins or of the chloroethyl group to either histones or acidic proteins was observed. Thus, the interaction of CCNU with the proteins of the cell nucleus can be defined in terms of the modification of histones by the cyclohexyl moiety. | Binding of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to L1210 cell nuclear proteins. The binding of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) to the proteins of the L1210 cell nucleus has been studied using both [cyclohexyl-14C]CCNU and [chloroethyl-14C]CCNU. Most of the bound [cyclohexyl-14C] moiety of CCNU was found to exist in a form that was stable in acid solution but labile and dialyzable in alkaline solution. A small amount of the cyclohexyl moiety was bound to histones in a stable, nondialyzable form. The drug/protein ratio for the H1 histone was about 0.01 to 0.02 mole/mole. No binding of the cyclohexyl group to acidic proteins or of the chloroethyl group to either histones or acidic proteins was observed. Thus, the interaction of CCNU with the proteins of the cell nucleus can be defined in terms of the modification of histones by the cyclohexyl moiety. | [
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PMID:4222 | 31P nuclear magnetic resonance-pH titrations of myo-inositol hexaphosphate. | With the use of 31P n.m.r. spectroscopy, the separate pKa values of each of the six phosphoric monoester groups of myo-inositol hexaphosphate were determined. The range of hydrogen-ion concentrations covered extended from that required for the phosphonium salts to that for the full dodecyl anion, and the determinations were carried out in the presence of sodium and tetrabutylammonium cations. The pKa for each phosphate grouping in the transition from the free acid forms of each group to the monoanion form of each group was determined to be: 1.1, C-2; 1.5, C-1 and C-3; 2.1, C-4 and C-6; and 1.7, C-5. In the mono- to di-anion transition, the pKa values were: 6.85, C-2; 7.60, C-5; 5.70 and 12.0, C-1 and C-3; and 10.0, C-4 and C-6. These data and the appearance of the 31P hexaphosphate n.m.r. multiplet are discussed in terms of conformations of myo-inositol hexaphosphate. | 31P nuclear magnetic resonance-pH titrations of myo-inositol hexaphosphate. With the use of 31P n.m.r. spectroscopy, the separate pKa values of each of the six phosphoric monoester groups of myo-inositol hexaphosphate were determined. The range of hydrogen-ion concentrations covered extended from that required for the phosphonium salts to that for the full dodecyl anion, and the determinations were carried out in the presence of sodium and tetrabutylammonium cations. The pKa for each phosphate grouping in the transition from the free acid forms of each group to the monoanion form of each group was determined to be: 1.1, C-2; 1.5, C-1 and C-3; 2.1, C-4 and C-6; and 1.7, C-5. In the mono- to di-anion transition, the pKa values were: 6.85, C-2; 7.60, C-5; 5.70 and 12.0, C-1 and C-3; and 10.0, C-4 and C-6. These data and the appearance of the 31P hexaphosphate n.m.r. multiplet are discussed in terms of conformations of myo-inositol hexaphosphate. | [
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PMID:4224 | Age dependence of the number of the stem cells in haemopoietic tissues of rats. | The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed. | Age dependence of the number of the stem cells in haemopoietic tissues of rats. The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed. | [
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PMID:4225 | Kinetics of haemopoietic recovery in endotoxin-treated mice. | Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 mug/blody weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells. Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time. | Kinetics of haemopoietic recovery in endotoxin-treated mice. Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 mug/blody weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells. Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time. | [
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PMID:4231 | [New interpretation of the symmetry of Cereus pedunculatus (Actiniaria) during embryonic development]. | A spatial reconstitution is used to determine with precision the relative position of pharynx and mesenteries during the embryonic development of Cereus pedunculatus. Three successive stages are described for embryonic symmetrisation. | [New interpretation of the symmetry of Cereus pedunculatus (Actiniaria) during embryonic development]. A spatial reconstitution is used to determine with precision the relative position of pharynx and mesenteries during the embryonic development of Cereus pedunculatus. Three successive stages are described for embryonic symmetrisation. | [
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PMID:4232 | [Effect of ligating the pancreatic duct on the intestinal pH in rabbits]. | In rabbits whose pancreatic ducts were ligatured, significant pH changes were observed in the anterior parts of the digestive tract (antrum and duodenum) but not in the posterior parts (jejunum and ileum) as could have been expected. These changes do not seem to be directly related to the lack of pancreatic juice, which leads one to think that, in the rabbit this secretion only plays a very limited role in the neutralisation processes. | [Effect of ligating the pancreatic duct on the intestinal pH in rabbits]. In rabbits whose pancreatic ducts were ligatured, significant pH changes were observed in the anterior parts of the digestive tract (antrum and duodenum) but not in the posterior parts (jejunum and ileum) as could have been expected. These changes do not seem to be directly related to the lack of pancreatic juice, which leads one to think that, in the rabbit this secretion only plays a very limited role in the neutralisation processes. | [
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PMID:4233 | [Demonstration of acidophilic neurosecretory cells in Crepidula fornicata Phil. (Mollusca, Gasteropoda, Prosobranchia) using the fluorescent dye, Geranine G]. | The Fluorochrome Geranine G binds to acidophilic neurosecretory cells and produces red orange fluorescence. Chromolipids and pigments show green fluorescence. This method is easy and sensitive. Fluorescence fading during irradiation is low. | [Demonstration of acidophilic neurosecretory cells in Crepidula fornicata Phil. (Mollusca, Gasteropoda, Prosobranchia) using the fluorescent dye, Geranine G]. The Fluorochrome Geranine G binds to acidophilic neurosecretory cells and produces red orange fluorescence. Chromolipids and pigments show green fluorescence. This method is easy and sensitive. Fluorescence fading during irradiation is low. | [
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PMID:4234 | [A glycoprotein: galactosyltransferase activity in the ascitic fluid and serum of mice with the YC8 tumor]. | Tranfer of (14C)-gal from exogenous UDP(14C)-gal has been demonstrated with ovomucoid as glycoprotein acceptor in ascitic fluid sera from Balb/c mice bearing isogenic YC8 tumor. Transfer exists without ovomucoid, in great ratio in ascitic fluid and in sera from ascitic mice too, showing occurrence of endogenous acceptors, the origin of which has to be proved. | [A glycoprotein: galactosyltransferase activity in the ascitic fluid and serum of mice with the YC8 tumor]. Tranfer of (14C)-gal from exogenous UDP(14C)-gal has been demonstrated with ovomucoid as glycoprotein acceptor in ascitic fluid sera from Balb/c mice bearing isogenic YC8 tumor. Transfer exists without ovomucoid, in great ratio in ascitic fluid and in sera from ascitic mice too, showing occurrence of endogenous acceptors, the origin of which has to be proved. | [
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PMID:4235 | Activity of gamma-glutamyl transpeptidase in serum of patients receiving anticonvulsant of anticoagulant therapy. | 1. Effects of chronic anticoagulant therapy in heart patients and anticonvulsant therapy in epileptics on gamma-glutamyl transpeptidase activity in serum were investigated. 2. The enzyme was elevated in 22% of 18 patients receiving anticoagulants. In these patients prothrombin time was also abnormally high. 3. 84% of 65 epileptics exhibited elevated gamma-glutamyl transpeptidase activity, 67% of which were not associated with elevated alkaline phosphatase or aspartate aminotransferase activities. In these latter cases, involvement of the liver was not apparent. 4. Possible relationships of anticonvulsant mediated enzyme induction or hepatic toxicity to elevated gamma-glutamyl transpeptidase activity in serum in epileptics is discussed. | Activity of gamma-glutamyl transpeptidase in serum of patients receiving anticonvulsant of anticoagulant therapy. 1. Effects of chronic anticoagulant therapy in heart patients and anticonvulsant therapy in epileptics on gamma-glutamyl transpeptidase activity in serum were investigated. 2. The enzyme was elevated in 22% of 18 patients receiving anticoagulants. In these patients prothrombin time was also abnormally high. 3. 84% of 65 epileptics exhibited elevated gamma-glutamyl transpeptidase activity, 67% of which were not associated with elevated alkaline phosphatase or aspartate aminotransferase activities. In these latter cases, involvement of the liver was not apparent. 4. Possible relationships of anticonvulsant mediated enzyme induction or hepatic toxicity to elevated gamma-glutamyl transpeptidase activity in serum in epileptics is discussed. | [
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PMID:4236 | Human saliva peroxidase: microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis. | Human saliva contains a high peroxidase activity that can be estimated spectrophotometrically with the hydrogen donor p-phenylenediamine and the substrate hydrogen peroxide from 20 mul of material. The pH optimum of the enzyme with citrate-phosphate buffer is 5.5. After microanalytical isoelectric fractionation 3 main isoenzyme components at pI 8.6, 6.5 and 4.3, and a number of isoenzyme subfractions at pI 9.5, 7.3 and 3.8 are detectable. In 3 patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt), in which a deficiency of leukocyte peroxidase had been reported by other authors, both the total activity of saliva peroxidase and the activity of individual isoenzymes were found to be within normal limits. These findings are not consistent with a generalized peroxidase deficiency in this disease. | Human saliva peroxidase: microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis. Human saliva contains a high peroxidase activity that can be estimated spectrophotometrically with the hydrogen donor p-phenylenediamine and the substrate hydrogen peroxide from 20 mul of material. The pH optimum of the enzyme with citrate-phosphate buffer is 5.5. After microanalytical isoelectric fractionation 3 main isoenzyme components at pI 8.6, 6.5 and 4.3, and a number of isoenzyme subfractions at pI 9.5, 7.3 and 3.8 are detectable. In 3 patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt), in which a deficiency of leukocyte peroxidase had been reported by other authors, both the total activity of saliva peroxidase and the activity of individual isoenzymes were found to be within normal limits. These findings are not consistent with a generalized peroxidase deficiency in this disease. | [
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PMID:4237 | Theoretical approaches to estimation of plasma renin activity: a review and some original observations. | Performance of accurrate, reproducible, and interpretable assays for plasma renin activity and other components of the renin/angiotensin system in the clinical setting requires a clear understanding of the various reactions in the renin/angiotensin cascade and the nature of their interactions. Plasma renin activity, the rate of angiotensin generations from plasma incubated in vitro, is the most commonly used clinical index of function in the renin/angiotensin system. Renin activity is measured by radioimmunoassay of angiotensin I generated in vitro under carefully controlled conditions. The value obtained for plasma renin activity depends on pH and duration of incubation and on the method used to protect the angiotensin I generated. We recommend incubation at neutral pH in buffered plasma for three hours in the presence of either ethylenediaminetetraacetate + 8-hydroxyquinolone + dimercaprol or ethylenediaminetetraacetate + phenylmethylsulfonylfluoride. Addition of a standard preparation of human renin to the plasma incubation step of the renin activity assay serves the dual purpose of permitting measurement of renin activity and furnishing an internal standard for comparison of assay procedures. The many variables among renin assay methods can be cancelled by referring to a common internal renin standard. | Theoretical approaches to estimation of plasma renin activity: a review and some original observations. Performance of accurrate, reproducible, and interpretable assays for plasma renin activity and other components of the renin/angiotensin system in the clinical setting requires a clear understanding of the various reactions in the renin/angiotensin cascade and the nature of their interactions. Plasma renin activity, the rate of angiotensin generations from plasma incubated in vitro, is the most commonly used clinical index of function in the renin/angiotensin system. Renin activity is measured by radioimmunoassay of angiotensin I generated in vitro under carefully controlled conditions. The value obtained for plasma renin activity depends on pH and duration of incubation and on the method used to protect the angiotensin I generated. We recommend incubation at neutral pH in buffered plasma for three hours in the presence of either ethylenediaminetetraacetate + 8-hydroxyquinolone + dimercaprol or ethylenediaminetetraacetate + phenylmethylsulfonylfluoride. Addition of a standard preparation of human renin to the plasma incubation step of the renin activity assay serves the dual purpose of permitting measurement of renin activity and furnishing an internal standard for comparison of assay procedures. The many variables among renin assay methods can be cancelled by referring to a common internal renin standard. | [
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PMID:4238 | Improved specificity of serum albumin determination and estimation of "acute phase reactants" by use of the bromcresol green reaction. | The reaction of serum samples with bromcresol green proceeds in two steps. Albumin is responsible for the faster (less than 1 min) reaction; the slower (30-min) reaction is a measure of "acute phase reactant(s)" in serum. Serum is simply mixed with bromcresol green reagent and the absorbance is measured twice, immediately and at 60 min. Albumin concentrations, determined from the absorbance at 0 min, correlate well with those determined by Laurell "rocket" immunoelectrophoresis; r = 0.95 with no certain deviation from unity for the slope and with a negligible difference at zero concentration. The slow reaction was expressed as deltaA% = 100 (deltaAs/deltaAv) where deltaAs and delta Av are the changes in absorbance between 60 and 0 min for the sample and a commercial control serum, respectively. The value for deltaA% correlates well with the percentage of alpha2-fraction as determined by electrophoresis on cellulose acetate, as well as with orosomucoid and ceruloplasmin, all of which are acute phase reactant(s). Whether these proteins or other acute phase reactant(s) actually cause the slow reaction has not yet been established. | Improved specificity of serum albumin determination and estimation of "acute phase reactants" by use of the bromcresol green reaction. The reaction of serum samples with bromcresol green proceeds in two steps. Albumin is responsible for the faster (less than 1 min) reaction; the slower (30-min) reaction is a measure of "acute phase reactant(s)" in serum. Serum is simply mixed with bromcresol green reagent and the absorbance is measured twice, immediately and at 60 min. Albumin concentrations, determined from the absorbance at 0 min, correlate well with those determined by Laurell "rocket" immunoelectrophoresis; r = 0.95 with no certain deviation from unity for the slope and with a negligible difference at zero concentration. The slow reaction was expressed as deltaA% = 100 (deltaAs/deltaAv) where deltaAs and delta Av are the changes in absorbance between 60 and 0 min for the sample and a commercial control serum, respectively. The value for deltaA% correlates well with the percentage of alpha2-fraction as determined by electrophoresis on cellulose acetate, as well as with orosomucoid and ceruloplasmin, all of which are acute phase reactant(s). Whether these proteins or other acute phase reactant(s) actually cause the slow reaction has not yet been established. | [
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PMID:4239 | Evaluation of lipase activity in serum by radial enzyme diffusion. | Results of determination of serum lipase by radial enzyme diffusion correlate well with those by a titrimetric reference method in the abnormal range. The specificity of the diffusion assay allows the differentiation of patients with pancreatic disease, even when the lipase activity of the serum is within the normal limits of the tritrimetric assay. Pancreatic lipase is not detectable by the diffusion assay in the serum of individuals who are free from pancreatic disease. | Evaluation of lipase activity in serum by radial enzyme diffusion. Results of determination of serum lipase by radial enzyme diffusion correlate well with those by a titrimetric reference method in the abnormal range. The specificity of the diffusion assay allows the differentiation of patients with pancreatic disease, even when the lipase activity of the serum is within the normal limits of the tritrimetric assay. Pancreatic lipase is not detectable by the diffusion assay in the serum of individuals who are free from pancreatic disease. | [
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PMID:4240 | Creatine kinase in serum: 1. Determination of optimum reaction conditions. | To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature. | Creatine kinase in serum: 1. Determination of optimum reaction conditions. To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature. | [
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PMID:4242 | Radioiodination of human low density lipoprotein: a comparison of four methods. | A comparison has been made of four labelling techniques used to radioiodinate human low density lipoprotein (LDL). (1) Chloramine T iodination at pH 7.4 was 20-25% efficient and gave a product immunologically indistinguishable from native LDL. Approximately 30% of the incorporated radioactivity, however, was found in LDL lipids, and the metabolic decay of the labelled complex in rats did not obey first order kinetics. Radiolabelling at pH 10 reduced the uptake of 125I into lipids to 10% but also cut the overall incorporation of radioiodine by a factor of 7. (2) Lactoperoxidase labelling and (3) conjugation with iodinated N-succinimidyl-3-(4-hydroxyphenyl)propionate were highly efficient (100%), but incorporation of radioactivity into the lipid moiety was unacceptably high (approximately 30%). (4) The efficiency of iodine monochloride labelling was highly reproducible and the product was immunologically indistinguishable from native LDL. Incorporation of radioactivity into the lipid moiety was less than 4% when the I/protein ratio of the product was kept at or below 1 : 1. Decay of the radiolabelled LDL in rats was monoexponential. | Radioiodination of human low density lipoprotein: a comparison of four methods. A comparison has been made of four labelling techniques used to radioiodinate human low density lipoprotein (LDL). (1) Chloramine T iodination at pH 7.4 was 20-25% efficient and gave a product immunologically indistinguishable from native LDL. Approximately 30% of the incorporated radioactivity, however, was found in LDL lipids, and the metabolic decay of the labelled complex in rats did not obey first order kinetics. Radiolabelling at pH 10 reduced the uptake of 125I into lipids to 10% but also cut the overall incorporation of radioiodine by a factor of 7. (2) Lactoperoxidase labelling and (3) conjugation with iodinated N-succinimidyl-3-(4-hydroxyphenyl)propionate were highly efficient (100%), but incorporation of radioactivity into the lipid moiety was unacceptably high (approximately 30%). (4) The efficiency of iodine monochloride labelling was highly reproducible and the product was immunologically indistinguishable from native LDL. Incorporation of radioactivity into the lipid moiety was less than 4% when the I/protein ratio of the product was kept at or below 1 : 1. Decay of the radiolabelled LDL in rats was monoexponential. | [
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PMID:4243 | Purification and properties of human acid-thermostable ribonucleases, and diagnosis of childhood pancreatic fibrosis. | Acid-thermostable ribonucleases were isolated from human pancreas, duodenal contents, liver, spleen, serum and urine, and purified 15--1000-fold. The pH optima, ionic requirements, and some of the specificity requirements, of these enzymes were investigated. The isolated enzymes formed two distinct groups: (a) The ribonucleases of the pancreas, duodenal contents and fraction A of serum and urine exhibit a pH optimum of 8.5, are inhibited by An2+ and Cu2+, and relatively rapidly hydrolyze the synthetic substrate uridine 3'-(alpha-naphthylphosphate); (b) the ribonucleases of the liver and spleen, and of fractions B of the serum and urine, with a pH optimum of 7, are less sensitive to An2+ and Cu2+, and exhibit negligible activity versus uridine 3'-(alpha-naphthylphosphate). Determination of the serum level of pancreatic-type ribonuclease activity, with the use of uridine 3'-(alpha-naphthylphosphate) or RNA as substrates, appears to be a valid diagnostic tool for pancreatic fibrosis in children. | Purification and properties of human acid-thermostable ribonucleases, and diagnosis of childhood pancreatic fibrosis. Acid-thermostable ribonucleases were isolated from human pancreas, duodenal contents, liver, spleen, serum and urine, and purified 15--1000-fold. The pH optima, ionic requirements, and some of the specificity requirements, of these enzymes were investigated. The isolated enzymes formed two distinct groups: (a) The ribonucleases of the pancreas, duodenal contents and fraction A of serum and urine exhibit a pH optimum of 8.5, are inhibited by An2+ and Cu2+, and relatively rapidly hydrolyze the synthetic substrate uridine 3'-(alpha-naphthylphosphate); (b) the ribonucleases of the liver and spleen, and of fractions B of the serum and urine, with a pH optimum of 7, are less sensitive to An2+ and Cu2+, and exhibit negligible activity versus uridine 3'-(alpha-naphthylphosphate). Determination of the serum level of pancreatic-type ribonuclease activity, with the use of uridine 3'-(alpha-naphthylphosphate) or RNA as substrates, appears to be a valid diagnostic tool for pancreatic fibrosis in children. | [
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PMID:4245 | Physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues in relation to glycogenosis type II (Pompe's disease). | The physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues have been studied. Heat stability of acid alpha-glucosidase from heart, liver and skeletal muscle is identical, but for kidney some different results are obtained. Identical isoelectrofocussing patterns are found for heart, liver and skeletal muscle. Furthermore, the effect of antiserum against human liver acid alpha-glucosidase on the activity of acid alpha-glucosidase from various tissues is studied. The results are discussed in relation to glycogenosis type II (Pompe's disease). | Physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues in relation to glycogenosis type II (Pompe's disease). The physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues have been studied. Heat stability of acid alpha-glucosidase from heart, liver and skeletal muscle is identical, but for kidney some different results are obtained. Identical isoelectrofocussing patterns are found for heart, liver and skeletal muscle. Furthermore, the effect of antiserum against human liver acid alpha-glucosidase on the activity of acid alpha-glucosidase from various tissues is studied. The results are discussed in relation to glycogenosis type II (Pompe's disease). | [
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PMID:4246 | Partial purification and properties of ovine liver Echinococcus granulosus protoscolices phospholgucose isomerase. | Echinococcus granulosus protoscolex is the actual larval stage of the cestode causing echinococcosis both in man and animals. In the present report, certain properties of phosphoglucose isomerase from the ovine liver E. granulosus protoscolices have been studied and compared with those of the hydatid cyst fluid and the healthy ovine liver enzymes. The protoscolices enzyme prepared in a manner similar to the hydatid cyst fluid and the ovine liver enzymes exhibited the following properties: (1) pH optimum of 8.2 (2) KM value of 0.23 mM, (3) the enzyme was inhibited in the presence of high concentrations of alpha-D-glucose 6-phosphate, (4) no detectable inhibition of the enzyme was observed in the presence of phosphate ion up to 4.1 mM, (5) the protoscolices enzyme was less thermostable as compared to the hydatid cyst fluid and the ovine liver enzymes, (6) the protoscolices enzyme had a lower Ki value (0.7 mM) as compared to either the hydatid cyst fluid (1.1 mM) or the ovine liver enzymes (4.6 mM) when 6-phosphogluconic acid was used as a competitive inhibitor. | Partial purification and properties of ovine liver Echinococcus granulosus protoscolices phospholgucose isomerase. Echinococcus granulosus protoscolex is the actual larval stage of the cestode causing echinococcosis both in man and animals. In the present report, certain properties of phosphoglucose isomerase from the ovine liver E. granulosus protoscolices have been studied and compared with those of the hydatid cyst fluid and the healthy ovine liver enzymes. The protoscolices enzyme prepared in a manner similar to the hydatid cyst fluid and the ovine liver enzymes exhibited the following properties: (1) pH optimum of 8.2 (2) KM value of 0.23 mM, (3) the enzyme was inhibited in the presence of high concentrations of alpha-D-glucose 6-phosphate, (4) no detectable inhibition of the enzyme was observed in the presence of phosphate ion up to 4.1 mM, (5) the protoscolices enzyme was less thermostable as compared to the hydatid cyst fluid and the ovine liver enzymes, (6) the protoscolices enzyme had a lower Ki value (0.7 mM) as compared to either the hydatid cyst fluid (1.1 mM) or the ovine liver enzymes (4.6 mM) when 6-phosphogluconic acid was used as a competitive inhibitor. | [
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PMID:4247 | Biochemical differences between the MB and MM isoenzymes of creatine kinase. | The biochemical properties of partially purified preparations of human myocardial MB and MM iosenzymes of creatine kinase have been compared with one another. Differences in substrate affinity, behaviour with inhibitors, and heat denaturation have been demonstrated. It is doubtful if the differences we have shown are sufficiently great to form the basis of a practical routine procedure for measuring serum levels of the MB isoenzyme. | Biochemical differences between the MB and MM isoenzymes of creatine kinase. The biochemical properties of partially purified preparations of human myocardial MB and MM iosenzymes of creatine kinase have been compared with one another. Differences in substrate affinity, behaviour with inhibitors, and heat denaturation have been demonstrated. It is doubtful if the differences we have shown are sufficiently great to form the basis of a practical routine procedure for measuring serum levels of the MB isoenzyme. | [
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PMID:4248 | The estimation of phospholipids in bile. | Total and lipid phosphorus were measured in the duodenal aspirate of 24 fasting subjects following an injection of cholecystokinin. The lipid phosphorus values were lower than the total phosphorus, a difference most pronounced in dilute samples. Storage at -20 degrees C over 4 weeks resulted in a loss of over 50% lipid phosphorus. Such alterations in the lipid phosphorus affected the calculation of biliary phospholipid and hence the saturation index of cholesterol in bile causing it to be erroneously elevated. It is concluded that analysis of bile samples should be undertaken on freshly obtained samples and include a preliminary step for the extraction of lipids. | The estimation of phospholipids in bile. Total and lipid phosphorus were measured in the duodenal aspirate of 24 fasting subjects following an injection of cholecystokinin. The lipid phosphorus values were lower than the total phosphorus, a difference most pronounced in dilute samples. Storage at -20 degrees C over 4 weeks resulted in a loss of over 50% lipid phosphorus. Such alterations in the lipid phosphorus affected the calculation of biliary phospholipid and hence the saturation index of cholesterol in bile causing it to be erroneously elevated. It is concluded that analysis of bile samples should be undertaken on freshly obtained samples and include a preliminary step for the extraction of lipids. | [
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PMID:4249 | Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease. | A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease. | Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease. A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease. | [
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PMID:4250 | X-linked ichthyosis, bilateral cryptorchidism, hypogenitalism and mental retardation in two siblings. | Two brothers showed ichthyosis, bilateral cryptorchidism, hypogenitalism and mental retardation. In addition, the younger brother had short stature associated with disorders of secretions of insulin, ACTH and GH. This is the third reported case of the syndrome of ichthyosis and hypogonadism. | X-linked ichthyosis, bilateral cryptorchidism, hypogenitalism and mental retardation in two siblings. Two brothers showed ichthyosis, bilateral cryptorchidism, hypogenitalism and mental retardation. In addition, the younger brother had short stature associated with disorders of secretions of insulin, ACTH and GH. This is the third reported case of the syndrome of ichthyosis and hypogonadism. | [
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PMID:4251 | A simple method for the cryopreservation of lymphocytes. Retention of specific immune effector functions by frozen-stored cells. | A simple, quick and inexpensive method for cryostorage of lymphocytes is discribed. When injected into appropriate normal recipients, frozen-stored rat and sheep lymphocytes caused GVH and NLT reactions respectively. Sheep lymphocytes remained viable after several months storage and functioned satisfactorily as 51Cr-labelled target cells in assays for cytotoxic antibodies. Lymphocytes, including immunoblasts, drained from sheep efferent lymphatics during immune responses to injected murine P815 cells or allogeneic lymphocytes survived freezing; when thawed the cells retained specific immune cytotoxic effector functions including the ability to secrete complement-dependent and leucocyte-dependent antibodies | A simple method for the cryopreservation of lymphocytes. Retention of specific immune effector functions by frozen-stored cells. A simple, quick and inexpensive method for cryostorage of lymphocytes is discribed. When injected into appropriate normal recipients, frozen-stored rat and sheep lymphocytes caused GVH and NLT reactions respectively. Sheep lymphocytes remained viable after several months storage and functioned satisfactorily as 51Cr-labelled target cells in assays for cytotoxic antibodies. Lymphocytes, including immunoblasts, drained from sheep efferent lymphatics during immune responses to injected murine P815 cells or allogeneic lymphocytes survived freezing; when thawed the cells retained specific immune cytotoxic effector functions including the ability to secrete complement-dependent and leucocyte-dependent antibodies | [
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PMID:4252 | Mechanism of post dialysis hyperventilation in patients with chronic renal insufficiency. | Several hypotheses have been put forward to explain postdialysis hypocapnia. Three were tested in this study: impairment of tissue oxygenation by dialysis (D)-induced alkalosis (Bohr effect), the D disequilibrium syndrome, and the loss of carbon dioxide (CO2) in D fluid. In 17 patients pre-DPCO2 was significantly correlated with plasma bicarbonate concentration (HCO3) and no disproportionate reduction of PCO2 was discernible. In 10 patients using a bath acetate concentration of 38 mEq/1 PCO2 was unchanged after D (35.4 versus 35.9 mm Hg before D), and was low relative to HCO3 whic increased from 21.2 to 28.0 mEq/1. After a dialysis using an acetate concentration of 25 mEq/1 HCO3 remained constant (20.4 versus 21.1 mEq/1 pre-D), whereas PCO2 fell from 35.3 to 30.8 mm Hg (P less than 0.001). Consequently PCO2 was again low relative to HCO3. Removal of CO2 by D fluid was excluded as a cause for low blood PCO2: addition of gaseous CO2 to the bath had no influence on arterial blood gases. Since post-D hypocapnia was not prevented when HCO3 was kept constant, it was concluded that post-D alkalosis cannot be the main reason for post-D hyperventilation, and that other factors related to the process of D are responsible. | Mechanism of post dialysis hyperventilation in patients with chronic renal insufficiency. Several hypotheses have been put forward to explain postdialysis hypocapnia. Three were tested in this study: impairment of tissue oxygenation by dialysis (D)-induced alkalosis (Bohr effect), the D disequilibrium syndrome, and the loss of carbon dioxide (CO2) in D fluid. In 17 patients pre-DPCO2 was significantly correlated with plasma bicarbonate concentration (HCO3) and no disproportionate reduction of PCO2 was discernible. In 10 patients using a bath acetate concentration of 38 mEq/1 PCO2 was unchanged after D (35.4 versus 35.9 mm Hg before D), and was low relative to HCO3 whic increased from 21.2 to 28.0 mEq/1. After a dialysis using an acetate concentration of 25 mEq/1 HCO3 remained constant (20.4 versus 21.1 mEq/1 pre-D), whereas PCO2 fell from 35.3 to 30.8 mm Hg (P less than 0.001). Consequently PCO2 was again low relative to HCO3. Removal of CO2 by D fluid was excluded as a cause for low blood PCO2: addition of gaseous CO2 to the bath had no influence on arterial blood gases. Since post-D hypocapnia was not prevented when HCO3 was kept constant, it was concluded that post-D alkalosis cannot be the main reason for post-D hyperventilation, and that other factors related to the process of D are responsible. | [
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PMID:4254 | Duration of cardiac effects of timolol and propranolol. | The duration of the cardiac effects of single intravenous doses of the beta-antagonists, timolol and propranolol, was compared in 6 healthy male subjects. Timolol and propranolol were given in doses of 1 mg and 10 mg, respectively, and at specified times after their administration, beta-blockade was assessed by the reduction of maximal exercise-induced tachycardia and by the inhibition of the chronotropic and inotropic effects of isoproterenol. Inotropic effects were measured by changes in the pre-ejection period of left ventricular systole obtained from systolic time intervals. There was no statistically significant difference in the timolol and propranolol time-courses of beta-blockade. The change in exercise-tachycardia was maximal 5 min after beta-antagonist infusion but dissipated rapidly so that no statistically significant change was observed 9 hr later. The chronotropic and inotropic effects of isoproterenol were almost completely antagonized for 11/2 hr after beta-antagonist infusion, and significant beta-blockade could be demonstrated 9 hr later. There was no difference in the time-course of the negative chronotropic and inotropic effects of either beta-antagonist. | Duration of cardiac effects of timolol and propranolol. The duration of the cardiac effects of single intravenous doses of the beta-antagonists, timolol and propranolol, was compared in 6 healthy male subjects. Timolol and propranolol were given in doses of 1 mg and 10 mg, respectively, and at specified times after their administration, beta-blockade was assessed by the reduction of maximal exercise-induced tachycardia and by the inhibition of the chronotropic and inotropic effects of isoproterenol. Inotropic effects were measured by changes in the pre-ejection period of left ventricular systole obtained from systolic time intervals. There was no statistically significant difference in the timolol and propranolol time-courses of beta-blockade. The change in exercise-tachycardia was maximal 5 min after beta-antagonist infusion but dissipated rapidly so that no statistically significant change was observed 9 hr later. The chronotropic and inotropic effects of isoproterenol were almost completely antagonized for 11/2 hr after beta-antagonist infusion, and significant beta-blockade could be demonstrated 9 hr later. There was no difference in the time-course of the negative chronotropic and inotropic effects of either beta-antagonist. | [
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PMID:4255 | Hypnotic efficacy of lorazepam and flurazepam. | In a double-blind crossover study involving 15 insomniac subjects, the hypnotic efficacy of lorazepam, 2 and 4 mg, was compared with flurazepam, 15 and 30 mg, and placebo. Five subjective measures were used: onset, length, and depth of sleep, number of times awakened, and satisfaction with the hypnotic. Lorazepam in 2- and 4-mg doses was comparable in hypnotic efficacy to flurazepam, 30 mg, according to most parameters of measurement. Side effects were minor, although relatively numerous at the 4-mg doses. | Hypnotic efficacy of lorazepam and flurazepam. In a double-blind crossover study involving 15 insomniac subjects, the hypnotic efficacy of lorazepam, 2 and 4 mg, was compared with flurazepam, 15 and 30 mg, and placebo. Five subjective measures were used: onset, length, and depth of sleep, number of times awakened, and satisfaction with the hypnotic. Lorazepam in 2- and 4-mg doses was comparable in hypnotic efficacy to flurazepam, 30 mg, according to most parameters of measurement. Side effects were minor, although relatively numerous at the 4-mg doses. | [
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PMID:4256 | Acebultolol: basis for the prediction of effect on exercise tolerance. | Twelve unselected males suffering from documented coronary insufficiency and moderately severe angina submitted to graded multistage treadmill exercise testing on 3 separate days, 3.5 hr after a single dose of 0,200, or 400 mg of acebulolol, a cardioselective beta blocker. Control measures included random allocation of 2 patients to each of 6 balanced sequences of administration, standardized double-blind conditions, and variance analysis for Latin-square design with repeated measures on each subject. Performance was evaluated by measuring time elapsed until anginal pain, peak heart rate, peak product of heart rate and blood pressure, and peak oxygen consumption. Mean values for all criteria were significantly atered by 400 mg of acebutolol. Seven out of twelve patients were classified as responders (i.e., exercise duration increased 100% or more). The response after acebutolol was correlated with the performance on placebo in the base of exercise duration, peak heart rate, and peak product of heart rate and blood pressure. It is concluded that: (1) performance criteria are useful predictors of response to beta blockade and (2) acebutolol is a potent antianginal agent when judged by an objective treadmill exercise test. | Acebultolol: basis for the prediction of effect on exercise tolerance. Twelve unselected males suffering from documented coronary insufficiency and moderately severe angina submitted to graded multistage treadmill exercise testing on 3 separate days, 3.5 hr after a single dose of 0,200, or 400 mg of acebulolol, a cardioselective beta blocker. Control measures included random allocation of 2 patients to each of 6 balanced sequences of administration, standardized double-blind conditions, and variance analysis for Latin-square design with repeated measures on each subject. Performance was evaluated by measuring time elapsed until anginal pain, peak heart rate, peak product of heart rate and blood pressure, and peak oxygen consumption. Mean values for all criteria were significantly atered by 400 mg of acebutolol. Seven out of twelve patients were classified as responders (i.e., exercise duration increased 100% or more). The response after acebutolol was correlated with the performance on placebo in the base of exercise duration, peak heart rate, and peak product of heart rate and blood pressure. It is concluded that: (1) performance criteria are useful predictors of response to beta blockade and (2) acebutolol is a potent antianginal agent when judged by an objective treadmill exercise test. | [
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PMID:4257 | Radiology in Mendelson's syndrome. | Four cases of Mendelson's syndrome (acid pulmonary aspiration) are presented. They all demonstrate an acute diffuse alveolar filling pattern. This appearance is by no means specific. However, in the absence of other causes for this pattern and of evidence of left ventricular failure the radiologist may alert the clinician to the correct diagnosis. Early recognition of this syndrome will result in prompt treatment which differs significantly from that of other causes of this radiographic appearance. The differential diagnosis is discussed. | Radiology in Mendelson's syndrome. Four cases of Mendelson's syndrome (acid pulmonary aspiration) are presented. They all demonstrate an acute diffuse alveolar filling pattern. This appearance is by no means specific. However, in the absence of other causes for this pattern and of evidence of left ventricular failure the radiologist may alert the clinician to the correct diagnosis. Early recognition of this syndrome will result in prompt treatment which differs significantly from that of other causes of this radiographic appearance. The differential diagnosis is discussed. | [
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PMID:4258 | Growth hormone secretion in acid-base alterations at rest and during exercise. | 1. Seven healthy males were studied during cycle ergometer exercise at 33%, 66% and 90% of VO2 max. on three occasions when NH4C1, NaHCO3 or CaCO3 (as a control substance) were administered in gelatin capsules double blind and in randomized order. Plasma growth hormone (HGH), lactic acid and hydrogen ion concentration ([H+]) were measured at frequent intervals. 2. Ammonium chloride produced highest blood [H+] and NaHCO3 the lowest. These differences were maintained during exercise and in recovery. Plasma lactic acid concentrations were similar at rest. At 66%, 90% VO2 max. and recovery lactic acid was highest with NaHCO3 and lowest with NH4C1. 3. Exercise stimulated HGH secretion in all studies and the elevation was proportional to the intensity of the exercise. NH4C1 caused a variable elevation of HGH at rest and 33% VO2 max. At 66% VO2 max., plasma HGH was significantly elevated to similar concentrations in all studies and, at 90% VO2 max., HGH was highest with NaHCO3. 4. An infusion of sodium L(+)-lactate producing plasma lactate concentrations of 3-5 mmol/l did not influence HGH secretion. 5. Exercise is a physiological stimulus to HGH secretion and the mechanism is independent of blood [H+] and lactate concentrations. | Growth hormone secretion in acid-base alterations at rest and during exercise. 1. Seven healthy males were studied during cycle ergometer exercise at 33%, 66% and 90% of VO2 max. on three occasions when NH4C1, NaHCO3 or CaCO3 (as a control substance) were administered in gelatin capsules double blind and in randomized order. Plasma growth hormone (HGH), lactic acid and hydrogen ion concentration ([H+]) were measured at frequent intervals. 2. Ammonium chloride produced highest blood [H+] and NaHCO3 the lowest. These differences were maintained during exercise and in recovery. Plasma lactic acid concentrations were similar at rest. At 66%, 90% VO2 max. and recovery lactic acid was highest with NaHCO3 and lowest with NH4C1. 3. Exercise stimulated HGH secretion in all studies and the elevation was proportional to the intensity of the exercise. NH4C1 caused a variable elevation of HGH at rest and 33% VO2 max. At 66% VO2 max., plasma HGH was significantly elevated to similar concentrations in all studies and, at 90% VO2 max., HGH was highest with NaHCO3. 4. An infusion of sodium L(+)-lactate producing plasma lactate concentrations of 3-5 mmol/l did not influence HGH secretion. 5. Exercise is a physiological stimulus to HGH secretion and the mechanism is independent of blood [H+] and lactate concentrations. | [
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PMID:4259 | The intracellular pH of human leucocytes in response to acid-base changes in vitro. | 1. Viable human leucocytes were isolated from venous blood and suspended in artificial media. Intracellular pH measurements were made by the dimethyloxazolidinedione technique in conditions simulating "respiratory" or "metabolic" acid-base disturbances. 2. Normal intracellular pH was 7-11 +/- 0-02 (mean +/- 2 SD) at an extracellular PCO2 of 5-8 kPa and a bicarbonate concentration of 25 mmol/l. 3. "Respiratory" and "metabolic" acidosis caused little change in pHi although increases in PCO2 led to relatively greater falls in pHi than did reduction in external bicarbonate concentration. 4. "Respiratory" and "metabolic" alkalosis caused similar and relatively greater increases in the pHi when compared with the response to an external acidosis. | The intracellular pH of human leucocytes in response to acid-base changes in vitro. 1. Viable human leucocytes were isolated from venous blood and suspended in artificial media. Intracellular pH measurements were made by the dimethyloxazolidinedione technique in conditions simulating "respiratory" or "metabolic" acid-base disturbances. 2. Normal intracellular pH was 7-11 +/- 0-02 (mean +/- 2 SD) at an extracellular PCO2 of 5-8 kPa and a bicarbonate concentration of 25 mmol/l. 3. "Respiratory" and "metabolic" acidosis caused little change in pHi although increases in PCO2 led to relatively greater falls in pHi than did reduction in external bicarbonate concentration. 4. "Respiratory" and "metabolic" alkalosis caused similar and relatively greater increases in the pHi when compared with the response to an external acidosis. | [
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PMID:4261 | Marrow transplantation in aplastic anemia and leukemia. | Human marrow transplantation has resulted in observations of fundamental significance in understanding both aplastic anemia and acute leukemia. For example, the observation that transplanted marrow can grow successfully in patients with aplastic anemia indicates that the disease is due to a defect in the marrow precursor cells and not in the marrow microenvironment. Similarly, the observation of recurrent leukemia in donor cells has important implications. Nonetheless, marrow transplantation is sufficiently established therapeutically to be considered the treatment of choice for patients with severe aplastic anemia, and a realistic alternative for patients with recurrent acute leukemia. We suggest that patients be managed with regard to marrow transplantation according to the general approach outlined in Table 3. Marrow transplantation and histocompatibility typing are available at increasing numbers of institutions throughout the world. More and more patients with either severe aplastic anemia or recurrent acute leukemia should have marrow transplantation available to them when it is indicated as part of optimal management of these no longer hopeless diseases. | Marrow transplantation in aplastic anemia and leukemia. Human marrow transplantation has resulted in observations of fundamental significance in understanding both aplastic anemia and acute leukemia. For example, the observation that transplanted marrow can grow successfully in patients with aplastic anemia indicates that the disease is due to a defect in the marrow precursor cells and not in the marrow microenvironment. Similarly, the observation of recurrent leukemia in donor cells has important implications. Nonetheless, marrow transplantation is sufficiently established therapeutically to be considered the treatment of choice for patients with severe aplastic anemia, and a realistic alternative for patients with recurrent acute leukemia. We suggest that patients be managed with regard to marrow transplantation according to the general approach outlined in Table 3. Marrow transplantation and histocompatibility typing are available at increasing numbers of institutions throughout the world. More and more patients with either severe aplastic anemia or recurrent acute leukemia should have marrow transplantation available to them when it is indicated as part of optimal management of these no longer hopeless diseases. | [
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PMID:4287 | The objective and timing of drug disposition studies, appendix V. A comparison of the bioavailability of three dosage forms of terfenadine. | Antagonistic effect against histamine-induced wheals was used to evaluate the bioavailability of terfenadine in monkeys. A rapidly dissolving tablet formulation of terfenadine shows essentially identical bioavailability to a liquid suspesion. A less readily dissolving capsule formulation lags considerably in time with regard to availability, and some question remains as to whether the total quantity in the capsule is available. | The objective and timing of drug disposition studies, appendix V. A comparison of the bioavailability of three dosage forms of terfenadine. Antagonistic effect against histamine-induced wheals was used to evaluate the bioavailability of terfenadine in monkeys. A rapidly dissolving tablet formulation of terfenadine shows essentially identical bioavailability to a liquid suspesion. A less readily dissolving capsule formulation lags considerably in time with regard to availability, and some question remains as to whether the total quantity in the capsule is available. | [
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PMID:4288 | The difficult hypertensive. | With the wide range of medications available today it should be possible to obtain satisfactory control in the majority of hypertensive patients. However, there are various categories of patients who may present particular problems in management, as for example patients with cerebro-vascular and coronary disease, or with renal failure. A particularly important group is those presenting with severe resistant hypertension, and these patients may constitute about 5 to 10% of the hypertensive population. Considerations relevant to the management of patients presenting with such problems are discussed. Combined drug regimens employing clonidine or beta-blockers with peripheral vasodilators appear to be particularly useful. | The difficult hypertensive. With the wide range of medications available today it should be possible to obtain satisfactory control in the majority of hypertensive patients. However, there are various categories of patients who may present particular problems in management, as for example patients with cerebro-vascular and coronary disease, or with renal failure. A particularly important group is those presenting with severe resistant hypertension, and these patients may constitute about 5 to 10% of the hypertensive population. Considerations relevant to the management of patients presenting with such problems are discussed. Combined drug regimens employing clonidine or beta-blockers with peripheral vasodilators appear to be particularly useful. | [
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PMID:4289 | [Our experience with the method of D'Amour and Smith]. | The authors carried out studies on a group of analgetic preparations (morphine, lydol, thylidine, pentazocine and analgine) by the method of D Amour and Smith, using thermic painful stimulation. The experiments were carried out on white rats-young and adult of both sexes of the strain Wistar. The mean lethal dosis were determined as well as the mean effective doses and therapeutic indices. The experimental results showed that thylidine was the strongest analgetic agent with atherapeutic index of 67. The index of morphine was 63 and was close to that of thylidine, but without statistical significance. The remaining analgetic indices were with low indices: lyndiol-with 5, pentazocine - 12 and analgine - 2. The method used, according to our data, could be applied successfully on rats of both sexes, at various age groups and both strains. The method is convenient for testing analgetic activity of preparations from two basic types of analgetic agents-narcotic and nonnarcotic. | [Our experience with the method of D'Amour and Smith]. The authors carried out studies on a group of analgetic preparations (morphine, lydol, thylidine, pentazocine and analgine) by the method of D Amour and Smith, using thermic painful stimulation. The experiments were carried out on white rats-young and adult of both sexes of the strain Wistar. The mean lethal dosis were determined as well as the mean effective doses and therapeutic indices. The experimental results showed that thylidine was the strongest analgetic agent with atherapeutic index of 67. The index of morphine was 63 and was close to that of thylidine, but without statistical significance. The remaining analgetic indices were with low indices: lyndiol-with 5, pentazocine - 12 and analgine - 2. The method used, according to our data, could be applied successfully on rats of both sexes, at various age groups and both strains. The method is convenient for testing analgetic activity of preparations from two basic types of analgetic agents-narcotic and nonnarcotic. | [
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PMID:4286 | Persisting and unaltered circadian rhythms of six healthy young men with a night-work shift every 48 hrs and a 2% CO2 atmosphere during a 4-week span. | Six apparently healthy young males (20 +/- 0.5 years of age) lived in a specially designed laboratory for a 1-week span in normal air, followed by 4 weeks in a 2% CO2 atmosphere and thereafter 1 week again in normal air. Room temperature was 24 degrees C. +/- 1 degrees C.; relative hygrometry 75% +/- 5%. With respect to socio-ecologic time clues and cues, the subjects were not isolated. The subjects' social synchronization was altered only by the shift-work schedule (light-on, 07(00); light-off, 22(30) on normal days). Every other day each subject had a 3-h night task, located between 23(00) and 07(00). Once a week, during 48 hrs (Saturday and Sunday) a set of physiologic variables was documented every 4 hrs in order to study their circadian changes: oral temperature, peak expiratory flow, grip strength, arterial blood pressure, tempo, and urinary pH, volume and potassium excretion. As far as rhythms are detectable (cosinor method) the most striking result is that both rhythm acrophases and amplitudes do not show any statistically significant changes when comparing either night-work versus day-work and/or normal air versus air with 2% CO2. Both 3 hrs of night-work every other day and an unusual amount of CO2 do not alter the parameters characterizing the circadian rhythms considered. The absence of desynchronization during night-work could be related to: 1) the speed of rotation in the shift-work; 2) the short duration of night-work; and 3) the youth of the subjects. | Persisting and unaltered circadian rhythms of six healthy young men with a night-work shift every 48 hrs and a 2% CO2 atmosphere during a 4-week span. Six apparently healthy young males (20 +/- 0.5 years of age) lived in a specially designed laboratory for a 1-week span in normal air, followed by 4 weeks in a 2% CO2 atmosphere and thereafter 1 week again in normal air. Room temperature was 24 degrees C. +/- 1 degrees C.; relative hygrometry 75% +/- 5%. With respect to socio-ecologic time clues and cues, the subjects were not isolated. The subjects' social synchronization was altered only by the shift-work schedule (light-on, 07(00); light-off, 22(30) on normal days). Every other day each subject had a 3-h night task, located between 23(00) and 07(00). Once a week, during 48 hrs (Saturday and Sunday) a set of physiologic variables was documented every 4 hrs in order to study their circadian changes: oral temperature, peak expiratory flow, grip strength, arterial blood pressure, tempo, and urinary pH, volume and potassium excretion. As far as rhythms are detectable (cosinor method) the most striking result is that both rhythm acrophases and amplitudes do not show any statistically significant changes when comparing either night-work versus day-work and/or normal air versus air with 2% CO2. Both 3 hrs of night-work every other day and an unusual amount of CO2 do not alter the parameters characterizing the circadian rhythms considered. The absence of desynchronization during night-work could be related to: 1) the speed of rotation in the shift-work; 2) the short duration of night-work; and 3) the youth of the subjects. | [
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PMID:4290 | [Blood sugar and hypoxic dynamics in metabolic acidosis and alkalosis (experimental data)]. | The dynamics of the glucose concentration and arterovenous glucose gradient was examined during different metabolic deviations in the acid-base balance. The metabolitic acidosis increased the level of sugar in the blood. A definite influence on the latter had the gravity of the acidosis and to a certain degree its pathogenetic form. The arterio-venous glucose difference changed from negative to positive with the increase of proton activity. The type of the peripheral hypoxic structure, formed during the state of acidosis influenced both the glucose gradient and its quantitative relations to the acid-base and oxygen indices. The sugar content in the blood during metabolitic alcalosis was not changed significantly. There was an increase in the arterio-venous gradient and inverse correlation between the glucose level and the arterio-venous oxygen difference with the increase of the bicarbonate concentration. | [Blood sugar and hypoxic dynamics in metabolic acidosis and alkalosis (experimental data)]. The dynamics of the glucose concentration and arterovenous glucose gradient was examined during different metabolic deviations in the acid-base balance. The metabolitic acidosis increased the level of sugar in the blood. A definite influence on the latter had the gravity of the acidosis and to a certain degree its pathogenetic form. The arterio-venous glucose difference changed from negative to positive with the increase of proton activity. The type of the peripheral hypoxic structure, formed during the state of acidosis influenced both the glucose gradient and its quantitative relations to the acid-base and oxygen indices. The sugar content in the blood during metabolitic alcalosis was not changed significantly. There was an increase in the arterio-venous gradient and inverse correlation between the glucose level and the arterio-venous oxygen difference with the increase of the bicarbonate concentration. | [
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PMID:4291 | [Effect of beta-adrenergic blockaders on the bioelectrical activity of the mesenchephalic reticular formation]. | The authors examined the beta-adrenergic structures in the mesencephalic formation (MSF) in view of establishing their role on the activity of MSF in view of establishing the in rols on the activity of MSF itself and the relationship of the same formation with the cerebral cortex and the lymbic system. It was established that beta-adrenergic blockers after their local application in MSF could inhibit its function. The data from the effect on the induced bioelectrical activity indicated also diminution in the functional activity of MSF. The authors found that the inhibiting influences of beta-blockers were abolished by their antagonist isoprenalite. An inference is made that there are beta-adrenergic structures, which act excitatory. | [Effect of beta-adrenergic blockaders on the bioelectrical activity of the mesenchephalic reticular formation]. The authors examined the beta-adrenergic structures in the mesencephalic formation (MSF) in view of establishing their role on the activity of MSF in view of establishing the in rols on the activity of MSF itself and the relationship of the same formation with the cerebral cortex and the lymbic system. It was established that beta-adrenergic blockers after their local application in MSF could inhibit its function. The data from the effect on the induced bioelectrical activity indicated also diminution in the functional activity of MSF. The authors found that the inhibiting influences of beta-blockers were abolished by their antagonist isoprenalite. An inference is made that there are beta-adrenergic structures, which act excitatory. | [
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PMID:4292 | [Age-related changes in the content and characteristics of glycosaminoglycans in human aortas]. | The vascular wall is rich in glucoseaminglicanes (GAG), which form the basic intercellular substance and determine its multiple functions. Using analytic and chromatographic methods the author examined hexauronic acid (HA), pentoses (P), sulphate groups and various derivatives of GAG (hyaluronic acid (HA), pentoses (P), sulphate groups and various derivatives of GAG (hyaluronic acid (HA), dermatan-sulfate (DS), keratansulfate (KS), chondroitinsulfate (HS), and cheransulfate (HES) in human aorta, obtained from persons at the age of 10 and over 70 years of age. Ten aortas of each group were examined. The studies showed that in the aortas of the adult persons the content of HA, P, HS, KS and sulphate groups was considerably higher than that of the aourtas, obtained form the young persons. There was an impression that the adult factor affected substantialy the qualitative characteristics of GAG--with advancement of age the lenght of polysaccharide chains was shortened and the degree of sulphatation was increased. The accumulation of acid GAG in the aorta of adult persons impaired the metabolism of substances between blood and tissue, which enhanced the infiltration of the blood vessels with lipids and proteins, especialy when there was a blood increase in their content (hyperlipemia, hypercholesterinemia, ect.) | [Age-related changes in the content and characteristics of glycosaminoglycans in human aortas]. The vascular wall is rich in glucoseaminglicanes (GAG), which form the basic intercellular substance and determine its multiple functions. Using analytic and chromatographic methods the author examined hexauronic acid (HA), pentoses (P), sulphate groups and various derivatives of GAG (hyaluronic acid (HA), pentoses (P), sulphate groups and various derivatives of GAG (hyaluronic acid (HA), dermatan-sulfate (DS), keratansulfate (KS), chondroitinsulfate (HS), and cheransulfate (HES) in human aorta, obtained from persons at the age of 10 and over 70 years of age. Ten aortas of each group were examined. The studies showed that in the aortas of the adult persons the content of HA, P, HS, KS and sulphate groups was considerably higher than that of the aourtas, obtained form the young persons. There was an impression that the adult factor affected substantialy the qualitative characteristics of GAG--with advancement of age the lenght of polysaccharide chains was shortened and the degree of sulphatation was increased. The accumulation of acid GAG in the aorta of adult persons impaired the metabolism of substances between blood and tissue, which enhanced the infiltration of the blood vessels with lipids and proteins, especialy when there was a blood increase in their content (hyperlipemia, hypercholesterinemia, ect.) | [
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PMID:4293 | [Beta blockers as psychotropic drugs]. | The range of indications for beta blocking substances has extended to various fields of pathology : vascular system, endocrinology... The discovery of some of their side effects give good reasons to try to find out whether they should have a place in psychopharmacotherapy and wheter they could meet its present requirements. The initial target symptoms were anxiety--which, being polymorphous, can be, at various degrees, reactionnal to obviously stressing surroundings, but which is also implied by structural distortions of personality--and psychosomatic manifestations. If their site of actions was initially thought peripherical, it now seems that their action is also central, genuinely psychotropic, anxiolytic, and non sedative, psychoanaleptic and, at the same time, stimulating and increasing the level of attention (we have observed in some cases a reduction of sleep time)--thymoanaleptic, increasing the incitement to action. On the other hand, they quiet certain excitation states caused by appearence of badly controled anxieties. This study contains three series of experiments conceived with a similar methodology. | [Beta blockers as psychotropic drugs]. The range of indications for beta blocking substances has extended to various fields of pathology : vascular system, endocrinology... The discovery of some of their side effects give good reasons to try to find out whether they should have a place in psychopharmacotherapy and wheter they could meet its present requirements. The initial target symptoms were anxiety--which, being polymorphous, can be, at various degrees, reactionnal to obviously stressing surroundings, but which is also implied by structural distortions of personality--and psychosomatic manifestations. If their site of actions was initially thought peripherical, it now seems that their action is also central, genuinely psychotropic, anxiolytic, and non sedative, psychoanaleptic and, at the same time, stimulating and increasing the level of attention (we have observed in some cases a reduction of sleep time)--thymoanaleptic, increasing the incitement to action. On the other hand, they quiet certain excitation states caused by appearence of badly controled anxieties. This study contains three series of experiments conceived with a similar methodology. | [
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PMID:4294 | Nuclear 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity for 5alpha-dihydrotestosterone in the rat prostate. | The in vitro examination of adult male rat prostatic 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity using 5alpha-dihydrotestosterone4 as substrate indicates that significant levels of enzyme activity are associated with purified nuclei as well as with the cytosol fractions. Both the purified nuclear and the cytosol fractions exhibited higher levels of 3alphaOHD activity with NADH than with NADPH. The pH activity curves for the NADH and NADPH catalyzed reactions were different for both the nuclear and cytosol fractions. The results suggest the presence of a number of 3alphaOHD enzymes in rat prostate. | Nuclear 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity for 5alpha-dihydrotestosterone in the rat prostate. The in vitro examination of adult male rat prostatic 3alpha-hydroxysteroid dehydrogenase (3alphaOHD) activity using 5alpha-dihydrotestosterone4 as substrate indicates that significant levels of enzyme activity are associated with purified nuclei as well as with the cytosol fractions. Both the purified nuclear and the cytosol fractions exhibited higher levels of 3alphaOHD activity with NADH than with NADPH. The pH activity curves for the NADH and NADPH catalyzed reactions were different for both the nuclear and cytosol fractions. The results suggest the presence of a number of 3alphaOHD enzymes in rat prostate. | [
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PMID:4295 | Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol. | Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO. | Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol. Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO. | [
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PMID:4296 | Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence. | The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions; luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH. The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine. | Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence. The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions; luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH. The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine. | [
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PMID:4297 | The effects of neurally active amino acids on prolactin secretion. | Several neurally active amino acids were injected into the third ventricle of anesthetized male rats. Two or eight mumole of GABA produced significant increases in the plasma concentrations of prolactin (PRL), indicating increased PRL release from the pituitary. Two mumole of glycine was also effective in elevating PRL levels. The intraventricular injection of the lowest dose of GABA (1.0 mumole), glutamate (0.4 or 2.3 mumole), lysine (0.2 or 2.0 mumole), or 0.9% NaCl did not alter PRL levels significantly. Plasma PRL concentrations did not increase following the injection of GABA or glycine directly into the anterior pituitary gland. The results suggest that GABA and glycine may play a role in the neural regulation of PRL secretion. | The effects of neurally active amino acids on prolactin secretion. Several neurally active amino acids were injected into the third ventricle of anesthetized male rats. Two or eight mumole of GABA produced significant increases in the plasma concentrations of prolactin (PRL), indicating increased PRL release from the pituitary. Two mumole of glycine was also effective in elevating PRL levels. The intraventricular injection of the lowest dose of GABA (1.0 mumole), glutamate (0.4 or 2.3 mumole), lysine (0.2 or 2.0 mumole), or 0.9% NaCl did not alter PRL levels significantly. Plasma PRL concentrations did not increase following the injection of GABA or glycine directly into the anterior pituitary gland. The results suggest that GABA and glycine may play a role in the neural regulation of PRL secretion. | [
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PMID:4298 | Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase. | A sensitive assay for d-3hydroxybutrate dehydrogenase (EC 1.1.1.30) was developed for use with the minute amounts of material obtained from islets of Langerhans microdissected from freeze-dried pancreatic sections. NADH formed in the enzyme reaction was determined by photokinetic analysis of the luminescence obtained with bacterial luciferase from Achromobacter fishcherii. In this way, accurate determination was obtained with less than 0.1 mug dry weight of islet material. In obese hyperglycemic mice, the islet enzyme had an activity of 4.7 mumoles/min and g dry weight. Optimal enzyme activity was found at pH 8 for the islet enzyme. The enzyme activity was similar in pancreatic islets and acini, while considerably higher activity was found in cardiac muscle, liver and renal cortex. Normal mouse islets showed about equal enzyme activity as the islets from obese hyperglycemic mice. | Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase. A sensitive assay for d-3hydroxybutrate dehydrogenase (EC 1.1.1.30) was developed for use with the minute amounts of material obtained from islets of Langerhans microdissected from freeze-dried pancreatic sections. NADH formed in the enzyme reaction was determined by photokinetic analysis of the luminescence obtained with bacterial luciferase from Achromobacter fishcherii. In this way, accurate determination was obtained with less than 0.1 mug dry weight of islet material. In obese hyperglycemic mice, the islet enzyme had an activity of 4.7 mumoles/min and g dry weight. Optimal enzyme activity was found at pH 8 for the islet enzyme. The enzyme activity was similar in pancreatic islets and acini, while considerably higher activity was found in cardiac muscle, liver and renal cortex. Normal mouse islets showed about equal enzyme activity as the islets from obese hyperglycemic mice. | [
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PMID:4299 | Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells. | Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs. By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P; these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes. | Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells. Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs. By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P; these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes. | [
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PMID:4300 | Clinical evaluation of blood lactate levels in equine colic. | Blood lactate levels were evaluated in 36 horses (43 cases) presented with colic. A correlation between increasing blood lactate levels and decreasing percentage survival has been shown. An appreciable anion gap was found in 7 of 10 cases analyzed in detail but in each case the entire gap could not be accounted for by lactate alone. Proposals are offered to account for the unmeasured anions. Blood lactate determination is suggested as a prognostic rather than a diagnostic aid for the equine practitioner and should be used to augment other clinical findings in the horse exhibiting colic. | Clinical evaluation of blood lactate levels in equine colic. Blood lactate levels were evaluated in 36 horses (43 cases) presented with colic. A correlation between increasing blood lactate levels and decreasing percentage survival has been shown. An appreciable anion gap was found in 7 of 10 cases analyzed in detail but in each case the entire gap could not be accounted for by lactate alone. Proposals are offered to account for the unmeasured anions. Blood lactate determination is suggested as a prognostic rather than a diagnostic aid for the equine practitioner and should be used to augment other clinical findings in the horse exhibiting colic. | [
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PMID:4301 | Equine viral encephalitis. | The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man. Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft. A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination. Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host". Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents. | Equine viral encephalitis. The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man. Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft. A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination. Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host". Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents. | [
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PMID:4302 | The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation. | Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds. | The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation. Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds. | [
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PMID:4303 | A calorimetric study of the CO Bohr effect of monomeric haemoglobins. | A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4 JK-1 mol-1) at pH 9.5 as compared to pH 6.0. | A calorimetric study of the CO Bohr effect of monomeric haemoglobins. A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4 JK-1 mol-1) at pH 9.5 as compared to pH 6.0. | [
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PMID:4304 | Conformational studies of two non-histone chromosomal proteins and their interactions with DNA. | The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA. | Conformational studies of two non-histone chromosomal proteins and their interactions with DNA. The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA. | [
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PMID:4305 | Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme. | 1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride. NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue. 4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity. | Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme. 1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride. NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue. 4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity. | [
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PMID:4306 | Membrane-reversible H+-ATPase from Micrococcus lysodeikticus. | Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase. | Membrane-reversible H+-ATPase from Micrococcus lysodeikticus. Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase. | [
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PMID:4307 | Active-site-directed inhibition of the plasma-membrane carrier transporting short-chain, neutral amino acids into Trypanosoma brucei. | 1. Glycine chloromethyl inhibited the active-transport of L-serine into bloodstream forms of Trypanosoma brucei. 2. Substrates of the short-chain, neutral amino acid transport system (N1), but not of other amino acid transport systems, protected the carrier protein from inhibition. 3. Inhibition was never more than 80% complete. The residual activity might have due to a proportion of N1 carrier active sites which had not reacted with the inhibitor. 4. The inhibition was highly selective for the N1 amino acid transport system. Other amino acid transport systems were not affected and the rate of respiration was only slightly affected. 5. The inhibition was first-order with respect to concentration, indicating that one molecule of the inhibitor reacted with each carrier active-site. 6. The high selectivity of this inhibitor should make it a useful labelling agent during the isolation and purification of the N1 amino acid transport carrier protein(s). | Active-site-directed inhibition of the plasma-membrane carrier transporting short-chain, neutral amino acids into Trypanosoma brucei. 1. Glycine chloromethyl inhibited the active-transport of L-serine into bloodstream forms of Trypanosoma brucei. 2. Substrates of the short-chain, neutral amino acid transport system (N1), but not of other amino acid transport systems, protected the carrier protein from inhibition. 3. Inhibition was never more than 80% complete. The residual activity might have due to a proportion of N1 carrier active sites which had not reacted with the inhibitor. 4. The inhibition was highly selective for the N1 amino acid transport system. Other amino acid transport systems were not affected and the rate of respiration was only slightly affected. 5. The inhibition was first-order with respect to concentration, indicating that one molecule of the inhibitor reacted with each carrier active-site. 6. The high selectivity of this inhibitor should make it a useful labelling agent during the isolation and purification of the N1 amino acid transport carrier protein(s). | [
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PMID:4308 | A quantitative model for partition in aqueous multiphase systems. | A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state. The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water. Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate. | A quantitative model for partition in aqueous multiphase systems. A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state. The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water. Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate. | [
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PMID:4309 | Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12. | When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan. | Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12. When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan. | [
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PMID:4310 | Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12. | Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5. | Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12. Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5. | [
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PMID:4311 | Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus. | Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption. | Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus. Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption. | [
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PMID:4312 | Multiple forms of cyclohexanone oxygenase from Nocardia globerula CL1. | The cyclohexanone 1,2-monooxygenase of Nocardia globerula CL1 exists as two electrophoretically distinct forms. These are present in crude cell extracts and are not artifacts of enzyme purification or electrophoresis. They have been separated in mg amounts by preparative polyacrylamide gel electrophoresis and shown to have essentially identical kinetic, spectral and physical characteristics. They do differ in pH-activity profile and temperature stability. Whether or not they are conformational isoenzymes or arise by gene duplication and divergent evolution has not been established. Cyclohexanone oxygenase constitutes 8% of the soluble protein of induced cells. This high level would correlate well with the presence of duplicate genes. It is proposed that the presence of a large amount of cyclohexanone oxygenase may confer an ecological advantage on the organism. | Multiple forms of cyclohexanone oxygenase from Nocardia globerula CL1. The cyclohexanone 1,2-monooxygenase of Nocardia globerula CL1 exists as two electrophoretically distinct forms. These are present in crude cell extracts and are not artifacts of enzyme purification or electrophoresis. They have been separated in mg amounts by preparative polyacrylamide gel electrophoresis and shown to have essentially identical kinetic, spectral and physical characteristics. They do differ in pH-activity profile and temperature stability. Whether or not they are conformational isoenzymes or arise by gene duplication and divergent evolution has not been established. Cyclohexanone oxygenase constitutes 8% of the soluble protein of induced cells. This high level would correlate well with the presence of duplicate genes. It is proposed that the presence of a large amount of cyclohexanone oxygenase may confer an ecological advantage on the organism. | [
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PMID:4313 | Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872. | 1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. | Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872. 1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. | [
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PMID:4314 | Purification and properties of a methanol-oxidizing enzyme in Pseudomonas C. | A methanol-oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion-exchange chromatography and gel filtration and resulted in a yield of 35.4%. Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures. The enzymes possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 muM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4. The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000. | Purification and properties of a methanol-oxidizing enzyme in Pseudomonas C. A methanol-oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion-exchange chromatography and gel filtration and resulted in a yield of 35.4%. Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures. The enzymes possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 muM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4. The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000. | [
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PMID:4315 | Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin. | The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature. The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule. | Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin. The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature. The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule. | [
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PMID:4316 | Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase. | The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum. | Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase. The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum. | [
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PMID:4317 | Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan. | To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time-consuming than techniques requiring hydrolysis or chemical reactions. | Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan. To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time-consuming than techniques requiring hydrolysis or chemical reactions. | [
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PMID:4318 | An extracellular aminopeptidase from Clostridium histolyticum. | An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate. | An extracellular aminopeptidase from Clostridium histolyticum. An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate. | [
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PMID:4319 | Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. | Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude. | Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude. | [
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PMID:4320 | Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. | Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed. | Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed. | [
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PMID:4321 | Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. | 1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form. | Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. 1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form. | [
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PMID:4322 | Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. | Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other. Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties. The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity. The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered. | Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other. Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties. The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity. The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered. | [
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PMID:4323 | Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. | 3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label. | Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. 3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label. | [
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PMID:4324 | Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. | The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given. | Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given. | [
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PMID:4325 | The proton electrochemical gradient in Escherichia coli cells. | The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required. | The proton electrochemical gradient in Escherichia coli cells. The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required. | [
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PMID:4326 | [Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]. | The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores... | [Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]. The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores... | [
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PMID:4327 | Comparison of the metal-ion-promoted dephosphorylation of the 5'-triphosphates of adenosine, inosine, guanosine and cytidine by Mn2+, Ni2+ and Zn2+ in binary and ternary complexes. | The dependence of the rate of dephosphorylation of ATP, ITP, GTP and CTP (= NTP), expressed as first-order rate constants (50 degrees C; I = 0.1 M, NaClO4), on pH (2 to 10), in the absence and presence of Mn2+, Ni2+, and Zn2+, was investigated. The reaction is accelerated by Zn2+ and passes through a pH optimum at about 8 for the system Zn2+-ATP or 9 for Zn2+-ITP and Zn2+-GTP; this is analogous to observations made earlier with the corresponding Cu2+ systems. By computing the pH dependence of the distribution of the several species present in these systems it is shown that the highest rates are observed in the pH regions where the concentration of Zn(ATP)2-, Zn(ITP-H)3-, or Zn(GTP-H)3- dominates. By evaluating the pH dependence evidence is given that the attacking nucleophile is OH- or H2O for Zn (ATP)2- and H2O for Zn (ITP-H)3- or Zn(GTP-H)3-. For all these complexes metal-ion/nucleic-base interactions are known, leading to the formation of macrochelates. These metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion-promoted dephosphorylation; in agreement with this, and the small tendency of the cytosine moiety to coordinate, the CTP systems are rather stable towards dephosphorylation. It should be noted that these experimental results do not necessarily mean that the macrochelates usually described are the reactive complexes, but only that the active complex must be closely related to them (e.g. isomers, etc). Although for the Ni2+ systems with ATP, ITP, and GTP, and for the Mn2+-ATP system a metal-ion/nucleic-base interaction is also known, these systems are not very sensitive to hydrolytic cleavage of the terminal P-O-P bond. The only known significant structural difference between the Ni2+-NTP or the Mn2+-ATP complexes and those of Cu2+ or Zn2+ is that Ni2+ Mn2+ coordinate to all three phsophate groups, whereas Cu2+ and Zn2+ involve only the beta and gamma ones. This structure-reactivity relationship is rationalized by the suggestion that in the active species the metal ion should be coordinated to the alpha,beta-phosphate groups leaving the gamma-group open to nucleophilic attack. Obviously, an initial beta,gamma-coordination is suitable for a shift of the metal ion along the phosphate back-bone into the reactive alpha-beta-position, while for an alpha,beta,gamma-coordination only the less favorable removal of the coordinated gamma-group remains. The metal-ion/nucleic-base interaction is considered as being important for achieving this reactive structure. The connection between trans-phosphorylation in vitro and in vivo is discussed. It is also shown that the formation of mixed-ligand or ternary complexes inhibits the dephosphorylation process. This is on the one hand of interest with regard to the transport of hydrolysis-sensitive phosphates in nature, while on the other it casts doubts on conclusions based on experiments carried out in the presence of buffers, because these contain weak bases and hence potential ligands. | Comparison of the metal-ion-promoted dephosphorylation of the 5'-triphosphates of adenosine, inosine, guanosine and cytidine by Mn2+, Ni2+ and Zn2+ in binary and ternary complexes. The dependence of the rate of dephosphorylation of ATP, ITP, GTP and CTP (= NTP), expressed as first-order rate constants (50 degrees C; I = 0.1 M, NaClO4), on pH (2 to 10), in the absence and presence of Mn2+, Ni2+, and Zn2+, was investigated. The reaction is accelerated by Zn2+ and passes through a pH optimum at about 8 for the system Zn2+-ATP or 9 for Zn2+-ITP and Zn2+-GTP; this is analogous to observations made earlier with the corresponding Cu2+ systems. By computing the pH dependence of the distribution of the several species present in these systems it is shown that the highest rates are observed in the pH regions where the concentration of Zn(ATP)2-, Zn(ITP-H)3-, or Zn(GTP-H)3- dominates. By evaluating the pH dependence evidence is given that the attacking nucleophile is OH- or H2O for Zn (ATP)2- and H2O for Zn (ITP-H)3- or Zn(GTP-H)3-. For all these complexes metal-ion/nucleic-base interactions are known, leading to the formation of macrochelates. These metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion-promoted dephosphorylation; in agreement with this, and the small tendency of the cytosine moiety to coordinate, the CTP systems are rather stable towards dephosphorylation. It should be noted that these experimental results do not necessarily mean that the macrochelates usually described are the reactive complexes, but only that the active complex must be closely related to them (e.g. isomers, etc). Although for the Ni2+ systems with ATP, ITP, and GTP, and for the Mn2+-ATP system a metal-ion/nucleic-base interaction is also known, these systems are not very sensitive to hydrolytic cleavage of the terminal P-O-P bond. The only known significant structural difference between the Ni2+-NTP or the Mn2+-ATP complexes and those of Cu2+ or Zn2+ is that Ni2+ Mn2+ coordinate to all three phsophate groups, whereas Cu2+ and Zn2+ involve only the beta and gamma ones. This structure-reactivity relationship is rationalized by the suggestion that in the active species the metal ion should be coordinated to the alpha,beta-phosphate groups leaving the gamma-group open to nucleophilic attack. Obviously, an initial beta,gamma-coordination is suitable for a shift of the metal ion along the phosphate back-bone into the reactive alpha-beta-position, while for an alpha,beta,gamma-coordination only the less favorable removal of the coordinated gamma-group remains. The metal-ion/nucleic-base interaction is considered as being important for achieving this reactive structure. The connection between trans-phosphorylation in vitro and in vivo is discussed. It is also shown that the formation of mixed-ligand or ternary complexes inhibits the dephosphorylation process. This is on the one hand of interest with regard to the transport of hydrolysis-sensitive phosphates in nature, while on the other it casts doubts on conclusions based on experiments carried out in the presence of buffers, because these contain weak bases and hence potential ligands. | [
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PMID:4328 | Rat-liver superoxide dismutase. Purification and age-related modifications. | Cytoplasmic superoxide dismutase has been purified from livers of young (6 months) and old (27 months) rats. The enzyme purified from old animals shows an age-related reduction in the specific activity, accumulation of antigenically cross-reacting material and increased sensitivity to temperature. No differences were found in the molecular weight, electrophoretic mobility, antigenicity and Ki between enzymes purified from young and old rats. This is the first demonstration of age-related alterations in a purified form of a non-metabolic enzyme, which can be related to reduced activity. The possible role of this reduced activity in age-dependent deterioration of cellular functions is discussed. | Rat-liver superoxide dismutase. Purification and age-related modifications. Cytoplasmic superoxide dismutase has been purified from livers of young (6 months) and old (27 months) rats. The enzyme purified from old animals shows an age-related reduction in the specific activity, accumulation of antigenically cross-reacting material and increased sensitivity to temperature. No differences were found in the molecular weight, electrophoretic mobility, antigenicity and Ki between enzymes purified from young and old rats. This is the first demonstration of age-related alterations in a purified form of a non-metabolic enzyme, which can be related to reduced activity. The possible role of this reduced activity in age-dependent deterioration of cellular functions is discussed. | [
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PMID:4330 | Effects of ouabain and hypoxia on the cardiac stimulation threshold in the dog (with special reference to changes in extra- and intracellular potassium concentrations). | The influence on the cardiac stimulation threshold of changes in the myocardial intra/extracellular potassium ratio induced with ouabain and hypoxia was investigated in 9 dogs with total atrioventricular block. In spite of significant elevations of extracellular potassium and reductions of intracellular potassium significant changes in stimulation threshold were not seen. It is suggested that different experimental conditions are responsible for the great variations in the results of previous studies of this subject. | Effects of ouabain and hypoxia on the cardiac stimulation threshold in the dog (with special reference to changes in extra- and intracellular potassium concentrations). The influence on the cardiac stimulation threshold of changes in the myocardial intra/extracellular potassium ratio induced with ouabain and hypoxia was investigated in 9 dogs with total atrioventricular block. In spite of significant elevations of extracellular potassium and reductions of intracellular potassium significant changes in stimulation threshold were not seen. It is suggested that different experimental conditions are responsible for the great variations in the results of previous studies of this subject. | [
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PMID:4331 | The effect of fructose infusions on the oxygen transport system of human blood. | In 9 healthy subjects the erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration, which modifies the oxygen affinity of haemoglobin, decreased by more than 25 per cent within 60 minutes of the beginning of a fructose infusion (0.5 g.kg body weight-1.h-1). In contrast erythrocyte adenosine triphosphate (ATP) concentration was unchanged. In spite of the diminished 2,3-DPG concentrations the oxygen affinity of whole blood actually measured remained unaltered. However, at the same time both the arterial and the venous blood pH had fallen by 0.05 or more. In vitro experiments indicated that this fall of erythrocyte 2,3-DPG was not due to a direct effect of fructose on the intra-erythrocytic regulation of 2,3-DPG or to changes indirectly related to the intravenous administration of fructose in vivo, i.e. an increase of the blood lactate/pyruvate ratio or a decrease of plasma inorganic phosphate. It is suggested that two opposing effects on the oxygen transport system of blood are induced by fructose infusions: 1) a displacement of the oxygen dissociation curve to the right due to the Bohr effect 2) a virtually counterbalancing shift of the oxygen dissociation curve to the left due to decreased erythrocyte 2,3-DPG concentrations. | The effect of fructose infusions on the oxygen transport system of human blood. In 9 healthy subjects the erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration, which modifies the oxygen affinity of haemoglobin, decreased by more than 25 per cent within 60 minutes of the beginning of a fructose infusion (0.5 g.kg body weight-1.h-1). In contrast erythrocyte adenosine triphosphate (ATP) concentration was unchanged. In spite of the diminished 2,3-DPG concentrations the oxygen affinity of whole blood actually measured remained unaltered. However, at the same time both the arterial and the venous blood pH had fallen by 0.05 or more. In vitro experiments indicated that this fall of erythrocyte 2,3-DPG was not due to a direct effect of fructose on the intra-erythrocytic regulation of 2,3-DPG or to changes indirectly related to the intravenous administration of fructose in vivo, i.e. an increase of the blood lactate/pyruvate ratio or a decrease of plasma inorganic phosphate. It is suggested that two opposing effects on the oxygen transport system of blood are induced by fructose infusions: 1) a displacement of the oxygen dissociation curve to the right due to the Bohr effect 2) a virtually counterbalancing shift of the oxygen dissociation curve to the left due to decreased erythrocyte 2,3-DPG concentrations. | [
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PMID:4332 | A comparison between a melanocyte-stimulating hormone inhibitory factor (MIF-I) and substances known to activate central dopamine receptors. | The tripeptide, prolyl-leucyl glycine amide, a melanocyte-stimulating hormone inhibitory factor (MIF-I), which has been reported to be effective in improving symptoms of Parkinson's disease, has been compared with drugs known to activate dopamine receptors in rat and mouse brain. Unlike apomorphine, amphetamine and amantadine it was incapable of producing sterotyped behaviour in the rat and unlike 1-dopa it was also ineffective in rats pretreated with the monoamineoxidase inhibitor mebanazine. Neither did it potentiate apomorphine nor amphetamine in this test. MIF-I did not antagonise chlorpromazine-induced loss of locomotor activity in mice, an effect which was antagonised by apomorphine, amphetamine and amantadine. Chlorpromazine hypothermia in the mouse was antagonised by 1-dopa but not by MIF-I; similar findings were obtained in reserpine-pretreated mice. These results suggest that the reported beneficial effect of MIF-I in Parkinson's disease is unlikely to be due to an interaction with dopamine systems in the brain. | A comparison between a melanocyte-stimulating hormone inhibitory factor (MIF-I) and substances known to activate central dopamine receptors. The tripeptide, prolyl-leucyl glycine amide, a melanocyte-stimulating hormone inhibitory factor (MIF-I), which has been reported to be effective in improving symptoms of Parkinson's disease, has been compared with drugs known to activate dopamine receptors in rat and mouse brain. Unlike apomorphine, amphetamine and amantadine it was incapable of producing sterotyped behaviour in the rat and unlike 1-dopa it was also ineffective in rats pretreated with the monoamineoxidase inhibitor mebanazine. Neither did it potentiate apomorphine nor amphetamine in this test. MIF-I did not antagonise chlorpromazine-induced loss of locomotor activity in mice, an effect which was antagonised by apomorphine, amphetamine and amantadine. Chlorpromazine hypothermia in the mouse was antagonised by 1-dopa but not by MIF-I; similar findings were obtained in reserpine-pretreated mice. These results suggest that the reported beneficial effect of MIF-I in Parkinson's disease is unlikely to be due to an interaction with dopamine systems in the brain. | [
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PMID:4334 | Acute graft-versus-host reaction in mice. 3. Organ distribution of injected 51 chromium labeled lymphocytes. | The distribution of labeled lymph node cells, causing an acute GvH reaction in lethally irradiated allogeneic recipients, was studied. Lymph node cells of C57BL mice were labeled with 51Cr and injected into lethally irradiated: a) C57BL mice, b) CBA mice, c) CBA mice sensitized to C57BL antigens prior to irradiation, d) CBA mice splenectomized before irradiation. Two more experimental situations were studied in which C57BL donors of lymph node cells were: e) presensitized to CBA antigens, or f) deprived of T-lymphocytes. The amount of radioactivity was determined in the whole body, blood, liver, spleen, subcutaneous lymph nodes, lungs, femora and kidneys of the irradiated recipient at regular intervals from the time of injection to the 120th hour after it. We found that living cells lodged predominantly in the spleen and the lymph nodes, while dead and dying cells accumulated in the liver. Other organs contained very small amounts of radioactivity. All the results point to the primary role of the spleen in the acute graft-versus-host reaction. | Acute graft-versus-host reaction in mice. 3. Organ distribution of injected 51 chromium labeled lymphocytes. The distribution of labeled lymph node cells, causing an acute GvH reaction in lethally irradiated allogeneic recipients, was studied. Lymph node cells of C57BL mice were labeled with 51Cr and injected into lethally irradiated: a) C57BL mice, b) CBA mice, c) CBA mice sensitized to C57BL antigens prior to irradiation, d) CBA mice splenectomized before irradiation. Two more experimental situations were studied in which C57BL donors of lymph node cells were: e) presensitized to CBA antigens, or f) deprived of T-lymphocytes. The amount of radioactivity was determined in the whole body, blood, liver, spleen, subcutaneous lymph nodes, lungs, femora and kidneys of the irradiated recipient at regular intervals from the time of injection to the 120th hour after it. We found that living cells lodged predominantly in the spleen and the lymph nodes, while dead and dying cells accumulated in the liver. Other organs contained very small amounts of radioactivity. All the results point to the primary role of the spleen in the acute graft-versus-host reaction. | [
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PMID:4335 | Mitigation of graft-versus-host disease in mice by treatment of donors with bacterial endotoxin. | Treatment of DBA/2 (H-2d) mice with bacterial endotoxin prior to transplantation of their spleen and lymph node cells into immunosuppressed AKR (H-2k) mice prevented acute mortality from graft-versus-host (GVH) disease. AKR mice that received immunocompetent cells from untreated DBA/2 mice had a median survival time (MST) of 13 days. In contrast, AKR mice that received immunocompetent cells from endotoxin-treated DBA/2 donors had an MST of 54 days. Endotoxin treatment of AKR recipients was not essential for preventing mortality from acute GVH disease. Chimerism was proved by demonstrating that the lymphoid cells of long-term surviving AKR mice had the characteristics of DBA/2 lymphoid cells as measured by their response in mixed leukocyte culture (MLC) tests. Spleen cells from endotoxin-treated DBA/2 mice were able to stimulate, and to be stimulated by, AKR spleen cells in MLC assays. Furthermore, spleen cells from endotoxin-treated DBA/2 mice did not suppress the responses of DBA/2 or AKR spleen cells in 'three-party' MLC tests. | Mitigation of graft-versus-host disease in mice by treatment of donors with bacterial endotoxin. Treatment of DBA/2 (H-2d) mice with bacterial endotoxin prior to transplantation of their spleen and lymph node cells into immunosuppressed AKR (H-2k) mice prevented acute mortality from graft-versus-host (GVH) disease. AKR mice that received immunocompetent cells from untreated DBA/2 mice had a median survival time (MST) of 13 days. In contrast, AKR mice that received immunocompetent cells from endotoxin-treated DBA/2 donors had an MST of 54 days. Endotoxin treatment of AKR recipients was not essential for preventing mortality from acute GVH disease. Chimerism was proved by demonstrating that the lymphoid cells of long-term surviving AKR mice had the characteristics of DBA/2 lymphoid cells as measured by their response in mixed leukocyte culture (MLC) tests. Spleen cells from endotoxin-treated DBA/2 mice were able to stimulate, and to be stimulated by, AKR spleen cells in MLC assays. Furthermore, spleen cells from endotoxin-treated DBA/2 mice did not suppress the responses of DBA/2 or AKR spleen cells in 'three-party' MLC tests. | [
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PMID:4341 | Influence of diurnal cycles on biochemical parameters of drug sensitivity: the pineal gland as a model. | Diurnal cycles in physical parameters in the environment modulate biochemical and physiological circadian rhythms in experimental animals, including cycles in the sensitivity to external influences. Environmental lighting synchronizes cycles of indole metabolism and melatonin synthesis in the rat pineal gland by modulating the activity of postganglionic sympathetic nerves. As a consequence, the sensitivity of pineal N-acetyltransferase to stimulation by isoproterenol or by dibutyryl cyclic AMP varies diurnally. Also, the capacity of actinomycin D to inhibit this induction varies with circadian periodicity. The cycles in sensitivity to isoproterenol reflect cycles in the system that regulates cyclic AMP production, and include variation in the availability of specific B-adrenergic binding sites, and in the sensitivity of receptor-coupled adenylate cyclase to catecholamines. Further, a variation in the response to dibutyryl cyclic AMP indicates in addition the participation of intracellular controls in the regulation of the sensitivity of N-acetyltransferase to catecholamines. The varying sensitivity to actinomycin D suggests a changing requirement for the synthesis of RNA as a function of prior environmental lighting conditions. The basic nature of these sensitivity changes suggests that diurnal cycles of environmental lighting may similarly affect other systems. | Influence of diurnal cycles on biochemical parameters of drug sensitivity: the pineal gland as a model. Diurnal cycles in physical parameters in the environment modulate biochemical and physiological circadian rhythms in experimental animals, including cycles in the sensitivity to external influences. Environmental lighting synchronizes cycles of indole metabolism and melatonin synthesis in the rat pineal gland by modulating the activity of postganglionic sympathetic nerves. As a consequence, the sensitivity of pineal N-acetyltransferase to stimulation by isoproterenol or by dibutyryl cyclic AMP varies diurnally. Also, the capacity of actinomycin D to inhibit this induction varies with circadian periodicity. The cycles in sensitivity to isoproterenol reflect cycles in the system that regulates cyclic AMP production, and include variation in the availability of specific B-adrenergic binding sites, and in the sensitivity of receptor-coupled adenylate cyclase to catecholamines. Further, a variation in the response to dibutyryl cyclic AMP indicates in addition the participation of intracellular controls in the regulation of the sensitivity of N-acetyltransferase to catecholamines. The varying sensitivity to actinomycin D suggests a changing requirement for the synthesis of RNA as a function of prior environmental lighting conditions. The basic nature of these sensitivity changes suggests that diurnal cycles of environmental lighting may similarly affect other systems. | [
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PMID:4342 | Nitrite, nitrosamines, and cancer. | Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution. | Nitrite, nitrosamines, and cancer. Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution. | [
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PMID:4345 | Hypothalamic inactivation of thyrotrophin-releasing hormone. | Following the demonstration of peptidases in the rat hypothalamus which inactivate thyrotrophin-releasing hormone (TRH), a sensitive and specific radioimmunoassay for the releasing hormone was used to investigate the presence of similar peptidases in the rabbit hypothalamus. TRH was found to be rapidly inactivated by supernatant and particulate hypothalamic fractions, with higher peptidase activity in the supernatant than in the particulate fraction. An optimum pH of 7.3 within physiological limits was obtained for the enzymes in both the fractions examined. The results obtained confirm that the rabbit hypothalamus contains enzymes capable of inactivating TRH, and since it has been found that such peptidases interfere with studies on TRH biosynthesis, it is possible that the peptidases may play a part in controlling the releasing hormone's production. The specificity of the antiserum used in the radioimmunoassay has also suggested that the peptidases may cleave the C-terminal-ProNH2,-NH2 or both from the TRH molecule to cause inactivation. | Hypothalamic inactivation of thyrotrophin-releasing hormone. Following the demonstration of peptidases in the rat hypothalamus which inactivate thyrotrophin-releasing hormone (TRH), a sensitive and specific radioimmunoassay for the releasing hormone was used to investigate the presence of similar peptidases in the rabbit hypothalamus. TRH was found to be rapidly inactivated by supernatant and particulate hypothalamic fractions, with higher peptidase activity in the supernatant than in the particulate fraction. An optimum pH of 7.3 within physiological limits was obtained for the enzymes in both the fractions examined. The results obtained confirm that the rabbit hypothalamus contains enzymes capable of inactivating TRH, and since it has been found that such peptidases interfere with studies on TRH biosynthesis, it is possible that the peptidases may play a part in controlling the releasing hormone's production. The specificity of the antiserum used in the radioimmunoassay has also suggested that the peptidases may cleave the C-terminal-ProNH2,-NH2 or both from the TRH molecule to cause inactivation. | [
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PMID:4361 | Effect of cimetidine on 24-hour intragastric acidity in normal subjects. | The effect of H2-receptor blockade on intragastric acidity was studied in nine normal males. The pH of their gastric contents was measured at hourly daytime and two hourly nighttime intervals for 48 hours. The subjects ate identical meals, drank identical volumes of fluid, and smoked the same number of cigarettes during the two study days. Their physical activity was unrestricted in a ward environment. Blood cimetidine and plasma gastrin were measured in serial blood samples. The nine subjects were treated in random sequence with cimetidine 0-8-1-0 g on one day and placebo capsules on the other. The drug was given in four divided doses: four subjects received it before, and five after, the three main meals. All took the fourth dose at bedtime. Replicate studies in an additional subject given placebo on both study days showed good reproducibility (r=0-80, P less than 0-01). Cimetidine therapy decreased intragastric acidity in all nine subjects. The decrease was similar in the two groups taking the drug before or after meals, mean 24 h intragastric hydrogen ion activity being lowered by 70 and 72% respectively. Nocturnal anacidity was recorded in only two of 45 samples. Administration of cimetidine before meals produced earlier and higher drug blood levels than post-prandial medication, but when it was taken after food the blood levels were highest at the time when the buffer capacity of the food was waning. Blood concentrations of cimetidine exceeded the secretory IC50 level for most of the time between doses. The results show that cimetidine 0-8-1-0 g/day in four divided doses produces a striking and consistent decrease of intragastric acidity. Although variation in the timing of the dose in relation to meals did not affect the decrease of acidity, the absorption data suggest that patients should take the drug after meals. | Effect of cimetidine on 24-hour intragastric acidity in normal subjects. The effect of H2-receptor blockade on intragastric acidity was studied in nine normal males. The pH of their gastric contents was measured at hourly daytime and two hourly nighttime intervals for 48 hours. The subjects ate identical meals, drank identical volumes of fluid, and smoked the same number of cigarettes during the two study days. Their physical activity was unrestricted in a ward environment. Blood cimetidine and plasma gastrin were measured in serial blood samples. The nine subjects were treated in random sequence with cimetidine 0-8-1-0 g on one day and placebo capsules on the other. The drug was given in four divided doses: four subjects received it before, and five after, the three main meals. All took the fourth dose at bedtime. Replicate studies in an additional subject given placebo on both study days showed good reproducibility (r=0-80, P less than 0-01). Cimetidine therapy decreased intragastric acidity in all nine subjects. The decrease was similar in the two groups taking the drug before or after meals, mean 24 h intragastric hydrogen ion activity being lowered by 70 and 72% respectively. Nocturnal anacidity was recorded in only two of 45 samples. Administration of cimetidine before meals produced earlier and higher drug blood levels than post-prandial medication, but when it was taken after food the blood levels were highest at the time when the buffer capacity of the food was waning. Blood concentrations of cimetidine exceeded the secretory IC50 level for most of the time between doses. The results show that cimetidine 0-8-1-0 g/day in four divided doses produces a striking and consistent decrease of intragastric acidity. Although variation in the timing of the dose in relation to meals did not affect the decrease of acidity, the absorption data suggest that patients should take the drug after meals. | [
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PMID:4366 | The effects of some factors on the growth and morphology of Naegleria sp. and three strains of the genus Acanthamoeba. | The effects of various biophysical and chemical factors on the cytology of vegetative stages of Naegleria sp., Vitek strain, Acanthamoeba culbertsoni, Acanthamoeba castellanii, Neff strain and Acanthamoeba polyphaga, No. 1289, were studied. The amoebae were cultured in a liquid medium under axenic conditions. The optimum temperature was 37 degrees C for pathogenic strains of Naegleria sp. and Acanthamoeba culbertsoni and 20 degrees C for A. castellanii. No changes were observed in the growth of A. polyphaga at the temperatures 20 degrees and 37 degrees C. The strains investigated grew at pH values of 5.6 to 7.7 using Soerensen's buffer. At the limit values the growth was inhibited and the morphology of cells was markedly changed. All of the four strains grew still at pH 8.4 kept by NaHCO3. A. polyphaga grew at partial anaerobiosis. The three tested strains of the genus Acanthamoeba grew in liquid axenic medium with 0.89% NaCl. The growth of Naegleria sp., Vitek was inhibited already at 0.2% concentration of this salt. The addition of 3 X 10(-2) m KCl to the culture medium had a harmful effect on the growth and morphology of three tested strains, except A. polyphaga. In the culture medium containing 2 X 10(-3) m CaCl2 the encystment of both pathogenic strains was stimulated. The cytological changes under experimental conditions were manifested by atypical movement of trophozoits and their intracellular structure. | The effects of some factors on the growth and morphology of Naegleria sp. and three strains of the genus Acanthamoeba. The effects of various biophysical and chemical factors on the cytology of vegetative stages of Naegleria sp., Vitek strain, Acanthamoeba culbertsoni, Acanthamoeba castellanii, Neff strain and Acanthamoeba polyphaga, No. 1289, were studied. The amoebae were cultured in a liquid medium under axenic conditions. The optimum temperature was 37 degrees C for pathogenic strains of Naegleria sp. and Acanthamoeba culbertsoni and 20 degrees C for A. castellanii. No changes were observed in the growth of A. polyphaga at the temperatures 20 degrees and 37 degrees C. The strains investigated grew at pH values of 5.6 to 7.7 using Soerensen's buffer. At the limit values the growth was inhibited and the morphology of cells was markedly changed. All of the four strains grew still at pH 8.4 kept by NaHCO3. A. polyphaga grew at partial anaerobiosis. The three tested strains of the genus Acanthamoeba grew in liquid axenic medium with 0.89% NaCl. The growth of Naegleria sp., Vitek was inhibited already at 0.2% concentration of this salt. The addition of 3 X 10(-2) m KCl to the culture medium had a harmful effect on the growth and morphology of three tested strains, except A. polyphaga. In the culture medium containing 2 X 10(-3) m CaCl2 the encystment of both pathogenic strains was stimulated. The cytological changes under experimental conditions were manifested by atypical movement of trophozoits and their intracellular structure. | [
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PMID:4369 | [Potassium substitutes during tocolysis]. | The level of serum potassium during tokolytic therapy with Partusisten and Isoptin decreases in the first 24 hours of therapy. This decrease is not due to an increase in renal potassium elimination. During tokolytic treatment the serum level of potassium returns to its normal value after 48 hours without potassium substitution. While there is a decrease in serum potassium noticeable changes in electrocardiogram which are typial for hypopotassemia are observed. These changes disappear afer 48 hours of tokolytic treatment but never the less the initial serum potassium drop should be balanced by potassium substitution within these 48 hours. | [Potassium substitutes during tocolysis]. The level of serum potassium during tokolytic therapy with Partusisten and Isoptin decreases in the first 24 hours of therapy. This decrease is not due to an increase in renal potassium elimination. During tokolytic treatment the serum level of potassium returns to its normal value after 48 hours without potassium substitution. While there is a decrease in serum potassium noticeable changes in electrocardiogram which are typial for hypopotassemia are observed. These changes disappear afer 48 hours of tokolytic treatment but never the less the initial serum potassium drop should be balanced by potassium substitution within these 48 hours. | [
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PMID:4370 | [Ambulatory therapy of trichomoniasis vaginalis. Clinical trial of a new Trichomonas-active azide substance]. | Report on the results of the clinical trial of a new trichomonacide in ambulatory patients in a gynaecological practice. A report is given on a new drug for the treatment of vaginal trichomoniasis, which has been tried in 87 patients of a gynaecological practice under outpatient conditions. The rate of failures was 5.7% (5 of 87 cases). The advantages over alternative preparations is to be seen in the short duration of treatment (24 hours at the most), and in the favourable tolerance in spite of high doses. | [Ambulatory therapy of trichomoniasis vaginalis. Clinical trial of a new Trichomonas-active azide substance]. Report on the results of the clinical trial of a new trichomonacide in ambulatory patients in a gynaecological practice. A report is given on a new drug for the treatment of vaginal trichomoniasis, which has been tried in 87 patients of a gynaecological practice under outpatient conditions. The rate of failures was 5.7% (5 of 87 cases). The advantages over alternative preparations is to be seen in the short duration of treatment (24 hours at the most), and in the favourable tolerance in spite of high doses. | [
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PMID:4375 | Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. | The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10- or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a Kav of between 0.4 and 0.6 and were further increased in potency by two- or fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mug/ml and that of the filtrate was less than 0.1 mug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of 5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin. | Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10- or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a Kav of between 0.4 and 0.6 and were further increased in potency by two- or fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mug/ml and that of the filtrate was less than 0.1 mug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of 5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin. | [
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PMID:4376 | Activity of two Streptococcus mutans bacteriocins in the presence of saliva, levan, and dextran. | The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology of the oral cavity. | Activity of two Streptococcus mutans bacteriocins in the presence of saliva, levan, and dextran. The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology of the oral cavity. | [
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